U.S. patent number 9,121,801 [Application Number 12/258,251] was granted by the patent office on 2015-09-01 for methods and devices for cellular analysis.
This patent grant is currently assigned to BioMarker Strategies, LLC. The grantee listed for this patent is Douglas P. Clark, Scott Diamond, Kathleen M. Murphy, Adam Schayowitz. Invention is credited to Douglas P. Clark, Scott Diamond, Kathleen M. Murphy, Adam Schayowitz.
United States Patent |
9,121,801 |
Clark , et al. |
September 1, 2015 |
Methods and devices for cellular analysis
Abstract
Embodiments of the present invention are directed to improved
methods and devices for analyzing a cell, aggregated cells, or a
solid tumor. Such methods and devices are, for example, useful in
the field of pathology and can provide improved cell processing and
analytical results.
Inventors: |
Clark; Douglas P. (Baltimore,
MD), Schayowitz; Adam (Bethesda, MD), Murphy; Kathleen
M. (Baltimore, MD), Diamond; Scott (Bala Cynwyd,
PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Clark; Douglas P.
Schayowitz; Adam
Murphy; Kathleen M.
Diamond; Scott |
Baltimore
Bethesda
Baltimore
Bala Cynwyd |
MD
MD
MD
PA |
US
US
US
US |
|
|
Assignee: |
BioMarker Strategies, LLC
(Rockville, MD)
|
Family
ID: |
40340698 |
Appl.
No.: |
12/258,251 |
Filed: |
October 24, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090162853 A1 |
Jun 25, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61099059 |
Sep 22, 2008 |
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60982279 |
Oct 24, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
23/12 (20130101); C12M 37/04 (20130101); G01N
15/1475 (20130101); C12M 23/42 (20130101); G01N
2035/1032 (20130101) |
Current International
Class: |
G01N
1/28 (20060101); G01N 15/14 (20060101); G01N
33/574 (20060101); G01N 1/30 (20060101); G01N
35/10 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 590 504 |
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Apr 1994 |
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EP |
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WO-93/24607 |
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Dec 1993 |
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WO |
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03/027236 |
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Apr 2003 |
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WO |
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2007/075440 |
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Jul 2007 |
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WO |
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Other References
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examiner .
Deepanwita Das, "Red Bllod Cell Stabilization: Effect on
Hydroxyethyl starch on RBC Viability, functionality and Oxidative
state during Freeze Thaw Conditions," eThesis, 2009, title page.
cited by examiner .
International Search Report for PCT/US2008/012148, issued May 26,
2009. cited by applicant .
Written Opinion for PCT/US2008/012148, mailed May 26, 2009. cited
by applicant .
Gdpawel, "Rare Cancer Support Forum", Jan. 1, 2006, XP055061044,
retrieved on Apr. 25, 2013. cited by applicant .
Holloway et al., "Association between in Vitro Platinum Resistance
in the EDR Assay and Clinical Outcomes for Ovarian Cancer
Patients", Gynecol. Oncol., 87(1):8-16 (2002). cited by applicant
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Krishnamurthy S., "Applications of molecular techniques to
fine-needle aspiration biopsy", Cancer (Cancer Cytopathology),
111(2):106-122 (2007). cited by applicant .
Schayowitz et al., "Functional profiling of live melanoma samples
using a novel automated platform", PLoS One, 7(12): e52760 (2012).
cited by applicant .
Weisenthal et al., "Platinum resistance determined by cell culture
drug resistance testing (CCDRT) predicts for patient survival in
ovarian cancer", pp. 1-25 (2003). Retrieved from the Internet: URL:
http://weisenthal.org/w.sub.--ovarian.sub.--cp.pdf. cited by
applicant .
Weisenthal L., "Functional Profiling for Targeted Drug Therapy with
Cell Culture Assays" (Nov. 20, 2006). Retrieved from the Internet:
URL:
http://www.weisenthal.org/Tokyo.sub.--Cancer.sub.--Symposium.sub.--Nov.su-
b.--2006.sub.--Weisenthal.pdf. cited by applicant .
European Patent Office communication from EP 08 841 971.8, dated
Sep. 9, 2013. cited by applicant.
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Primary Examiner: Yakovleva; Galina
Attorney, Agent or Firm: DLA Piper LLP (US)
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional application
61/099,059 filed Sep. 22, 2008 and U.S. provisional application
60/982,279 filed Oct. 24, 2007, the subject matter of both is
incorporated herein by reference in its entirety.
Claims
We claim:
1. A method for processing cancer cells from a solid tumor sample
from a subject comprising the following steps in sequential order:
(a) disaggregating and dispersing an aqueous solution containing
live aggregated cancer cells into at least one test aliquot in a
first isolated chamber, wherein a predetermined amount of laminar
fluid shear force to disrupt aggregates of the cancer cells without
killing the cancer cells and triggering only a minimal stress
response or no stress response in the cancer cells is used; (b)
optionally purifying the aliquot to increase the percentage of
target cancer cells relative to other contaminating cell types by
removing the contaminating cells; (c) distributing the optionally
purified live cancer cells into one or more second isolated
chambers for analysis; (d) contacting the distributed live cancer
cells ex vivo with at least one agent to produce a measurable
quantitatively or qualitative effect on a target ex vivo biomarker
or biomolecules in a cellular pathway; (e) stabilizing the target
ex vivo biomarker or biomolecule of the cancer cells within about
one to four hours by lysing or fixing the cancer cells on a solid
support using a solution comprising a polymer, thereby killing the
cancer cells; and (f) measuring the changes in levels of the target
ex vivo biomarker or biomolecule in the cellular pathway to assess
the response of the target cancer cells to the at least one
agent.
2. The method of claim 1, wherein the subject is a human and the
method is performed at the point of care.
3. The method of claim 1, wherein the cancer cells are obtained
from the subject as a solid tumor biopsy.
4. The method of claim 1, wherein the solid tumor sample is
obtained using a fine needle aspiration technique.
5. The method of claim 1, in which the total number of the live
aggregated cancer cells processed is between about 1000 and
10.times.10.sup.6.
6. The method of claim 1, wherein the disaggregation step comprises
passing the fluid comprising the cancer cells from the solid tumor
sample through a needle or pipette tip of a predetermined size.
7. The method of claim 1, wherein the cancer cells are dispersed by
the shear force of between about 100 to about 800
dyne/cm.sup.2.
8. The method of claim 1, wherein the optional purification
comprises immunodepletion.
9. The method of claim 1, wherein the one or more second isolated
chambers contain less than about 1,000,000 of the purified cancer
cells.
10. The method of claim 1, wherein the distribution step is done
manually or using an automated system.
11. The method of claim 1, wherein the distributed live cancer
cells have over about 75% viability as compared to the number of
viable cancer cells in the fluid prior to the distribution.
12. The method of claim 1, wherein the distributed live cancer
cells are divided into at least two aliquots and wherein each
aliquot is contacted with a different at least one agent in step
(d).
13. The method of claim 1, wherein the at least one agent is
selected from the group consisting of a pharmaceutical agent, an
agent for stimulating a cell, a polypeptide, a polynucleotide, an
antibody, an Fab fragment, an Fc fragment, RNA, siRNA and a
phosphoprotein.
14. The method of claim 1, wherein the cellular pathway is selected
from the group consisting of a metabolic pathway, a replication
pathway, a cellular signaling pathway, an oncogenic signaling
pathway, an apoptotic pathway, and a pro-angiogenic pathway.
15. The method of claim 1, wherein the agent is an epidermal growth
factor (EGF).
16. The method of claim 1, wherein the quantitative or qualitative
effect measured is the expression level of a gene selected from the
group consisting of an immediate or delayed early gene family.
17. The method of claim 1, wherein the target ex vivo biomarker or
biomolecule is selected from the group consisting of ions, enzymes,
lipids, and post-translationally modified proteins.
18. The method of claim 13, wherein the at least one agent is
preloaded into one or more second isolated chambers before the
purified live cancer cells are distributed into the one or more
second isolated chambers.
19. The method of claim 1, wherein the at least one agent is a
detectable agent selected from the group consisting of: an enzyme,
fluorescent material, luminescent material, bioluminescent
material, radioactive material, positron emitting metal using a
positron emission tomography, and nonradioactive paramagnetic metal
ion.
20. The method of claim 1, wherein the solid support is a glass
slide.
Description
FIELD OF THE INVENTION
Embodiments of the present invention are directed to improved
methods and devices for analyzing a cell, aggregated cells, or a
solid tumor. Such methods and devices are, for example, useful in
the field of pathology and can provide improved cell processing and
analytical results.
BACKGROUND
Traditional pathological samples have been largely processed using
methods that involve killing the cells or lengthy sample processing
times. Such methods are generally performed in a laboratory well
away from the point of care. These traditional methods do not
permit the examination of live cells, including dynamic, live-cell
related biomarkers, and do not allow for rapid sample processing or
analytical result generation at the point of care. This lack of
complete and rapidly obtained information can prevent doctors from
identifying the proper treatment regimen or at the least slow the
process which adversely effects the patient's quality of life. A
comparison of the traditional process to some improved embodiments
is shown in FIG. 9.
For example, oncologists have a number of treatment options
available to them, including different combinations of drugs that
are characterized as standard of care, and a number of drugs that
do not carry a label claim for a particular cancer, but for which
there is evidence of efficacy in that cancer. The best likelihood
of good treatment outcome requires that patients be assigned to
optimal available cancer treatment, and that this assignment be
made as quickly as possible following diagnosis.
While some cancers can be readily identified using genomic markers,
reliable genomic markers are not available for all cancers, which
may be better characterized as exhibiting abnormal expression of
one or (typically) many normal genes. Currently available
diagnostic tests to diagnose particular types of cancer and
evaluate the likely effectiveness of different treatment strategies
based on gene expression may have one or more disadvantages, for
example: (1) the tests may be designed for testing blood and are
not readily adapted for testing solid tumors; (2) sample
preparation methods for solid tumor samples, including
disaggregation of cells, may be unsuitable for handling live cells
or performing subsequent measurements of marker expression; (3)
small samples, e.g., obtained using fine needle biopsies, may not
provide sufficient tissue for complete analysis; (4) the tests may
require in vitro culturing of the cells, extended incubation
periods, and/or significant delays between the time that the test
cells are obtained from the patient and the time the cells are
tested, resulting potential for wide variation and external
influences on marker expression; (5) the tests may be unsuited for
measuring expression of a multiplicity of genes, phosphoproteins or
other markers in parallel, which may be critical for recognizing
and characterizing the expression as abnormal; (6) the tests may be
non-quantitative, relying principally on immunohistochemistry to
determine the presence or absence of a protein as opposed to
relative levels of expression of genes; (7) the reagents and cell
handling conditions are not strictly controlled, leading to a high
degree of variability from test to test and lab to lab; (8) the
tests may be unsuited to analyzing RNA levels, due to the
instability of RNA and the practical difficulty of obtaining
sufficiently fresh samples from the patients; and (9) the tests may
involve fixing of the cells before any gene expression analysis can
be performed, e.g., in the presence or absence of selected
reagents.
Recently, several groups have published studies concerning the
classification of various cancer types by microarray gene
expression analysis (see, e.g. Golub et al., Science 286:531-537
(1999); Bhattacharjae et al., Proc. Nat. Acad. Sci. USA
98:13790-13795 (2001); Chen-Hsiang et al., Bioinformatics 17
(Suppl. 1): S316-S322 (2001); Ramaswamy et al., Proc. Natl. Acad.
Sci. USA 98:1514915154 (2001)). Certain classifications of human
breast cancers based on gene expression patterns have also been
reported (Martin et al., Cancer Res. 60:2232-2238 (2000); West et
al., Proc. Natl. Acad. Sci. USA 98:11462-11467 (2001); Sorlie et
al., Proc. Natl. Acad. Sci. USA 98:1086910874 (2001); Yan et al.,
Cancer Res. 61:8375-8380 (2001)). However, these studies mostly
focus on improving and refining the already established
classification of various types of cancer, including breast cancer,
and generally do not provide new insights into the relationships of
the differentially expressed genes. These studies do not link the
findings to treatment strategies in order to improve the clinical
outcome of cancer therapy, and they do not address the problem of
improving and standardizing existing techniques of cell handling
and analysis.
Although modern molecular biology and biochemistry have revealed
more than 100 genes whose activities influence the behavior of
tumor cells, state of their differentiation, and their sensitivity
or resistance to certain therapeutic drugs, with a few exceptions,
the status of these genes has not been exploited for the purpose of
routinely making clinical decisions about drug treatments. One
notable exception is the use of estrogen receptor (ER) protein
expression in breast carcinomas to select patients to treatment
with anti-estrogen drugs, such as tamoxifen. Another exceptional
example is the use of ErbB2 (Her2) protein expression in breast
carcinomas to select patients with the Her2 antagonist drug
Herceptin.RTM. (Genentech, Inc., South San Francisco, Calif.). For
most cancers, however, the pathologies in gene expression may be
subtler and may involve patterns of expression of multiple genes or
expression of genes in response to particular stimuli.
The challenge of cancer treatment remains to target specific
treatment regimens to pathogenically distinct tumor types, and to
identify the optimal treatment as early as possible in order to
optimize outcome. Hence, a need exists for tests that
simultaneously provide prognostic and/or predictive information
about patient responses to the variety of treatment options.
There is a need for a device and a method to prepare solid tumor
biopsies or otherwise aggregated cells which address these
disadvantages and integrate, in a single small and compact
apparatus, the function of handling and preparing tissue samples
using controlled, consistent and efficient steps; maintaining
viability of the tissue sample, to permit stimulation and/or
preservation of different biomarker responses from the same tissue
sample before the sample loses viability or becomes cultured
through ex vivo replication.
SUMMARY
Embodiments of the present invention are directed to methods for
processing or preparing a live tissue sample of aggregated cells
from a subject. These methods can include: disaggregating and
dispersing an aqueous solution containing live aggregated cells
obtained from a subject into at least one test aliquot in a first
isolated chamber; optionally purifying the aliquot to increase the
percentage of target cells relative to other contaminating cell
types by removing the contaminating cells; distributing the
optionally purified live cells into one or more second isolated
chambers for analysis; and stabilizing the distributed cells to
permit cellular and/or molecular analysis of the distributed
cells.
The present invention is also directed to, in some embodiments,
methods for processing or preparing cancer cells from a solid tumor
that include: disaggregating and dispersing live cancer cells
obtained from a solid tumor into at least one test aliquot in at
least one first isolated chamber; optionally purifying the live
cancer cells to remove contaminants; distributing the live cancer
cells into one or more second isolated chambers for analysis; and
stabilizing the distributed cells to permit cellular and/or
molecular analysis of the cells.
Further, some embodiments of the present invention are directed to
cartridges for cellular processing. For example, cartridges for use
in processing or preparing live cancer cells having a plurality of
sterile compartments, wherein the compartments can be separated
from one another. Other embodiments are also directed to cartridges
having a plurality of compartments including: a compartment for
dispersing cells, a compartment for purifying cells, and a
compartment that is an isolated chamber.
The invention is also directed to systems including a cartridge of
the present invention and an analytical device. Kits including the
cartridges of the present invention are also encompassed
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an example embodiment of a cartridge of the present
invention.
FIG. 2 shows exemplary isolated chambers within a cartridge for
receiving and handling a sample.
FIG. 3 shows an example disaggregation process according to the
invention.
FIG. 4 shows an additional embodiment of a cartridge of the present
invention.
FIG. 5 shows a cross section and top view of an exemplary
embodiment of a glass slide holder that can be positioned on a
cartridge of the present invention.
FIG. 6 shows an additional embodiment of a cartridge of the present
invention illustrating exemplary features.
FIG. 7 shows an example view of an embodiment of the cartridge of
the present invention where some of the compartments of the
cartridge can be separated from one another.
FIG. 8 shows an additional embodiment of a cartridge of the present
invention illustrating exemplary features.
FIG. 9 shows a comparison of a traditional pathology sample
processing method (left) with an example processing method of the
present invention (right).
FIG. 10 shows that the methods of the present invention produce RNA
samples having a higher RNA integrity number (RIN) than a formalin
fixation paraffin embedded process.
FIG. 11 provides an illustration showing that traditional tumor
sample processing methods can damage biomarkers which can reduce
the cellular information available.
FIG. 12 shows an example processing method of the present invention
that begins with extraction using fine needle aspiration (FNA).
FIG. 13 shows RIN scores 13A, .mu.g of RNA produced 13B, and
260/280 values 13C in HCT-116 cells at varying cell numbers.
FIG. 14 shows RIN scores 14A, .mu.g of RNA produced 14B, and
260/280 values 14C in MCF-7 cells at varying cell numbers.
FIG. 15 shows the impact of dispersion at varying dyne/cm.sup.2 on
cluster size in MCF-7 and HCT-116 cells.
FIG. 16 shows the impact of dispersion at varying dyne/cm.sup.2 on
cell viability in MCF-7 and HCT-116 cells.
FIG. 17 shows varying levels of FOS induction from dispersion and
EGF stimulation in MCF-7 cells.
FIG. 18 shows varying levels of FOS induction from dispersion and
EGF stimulation in HCT-116 cells.
FIG. 19 shows tumor cell enrichment in MCF-7 and HCT-116 cells
using the methods of the present invention.
FIG. 20 shows the results of using live cell probes in MCF-7 cells
prepared using the present methods versus those prepared using a
three hour formalin fixation procedure.
FIG. 21 shows the results of using live cell probes in HCT-116
cells prepared using the present methods versus those prepared
using a three hour formalin fixation procedure.
FIG. 22 shows an example dispersion of MCF-7 and HCT-116 cells
using the disaggregation techniques described herein.
DETAILED DESCRIPTION
Embodiments of the invention described herein include, but are not
limited to, an automated, self-contained, fluidic tumor cell
processing and testing system that promotes the development and use
of targeted therapies and molecular diagnostic tests. Embodiments
of this invention also include methods of using the system,
including improved pathological processing methods that can be
performed on live cells ex vivo. The present invention is also
directed to kits for use with the system and methods described
herein.
The invention provides a safe, effective, accurate, precise,
reproducible, inexpensive, cost effective, efficient, fast and
convenient method and "cartridge-based" system for collecting,
handling and processing of solid cellular specimens ex vivo. These
methods and cartridges can maintain viability of the samples during
the process to maintain biomarker integrity, and optionally,
evoking biomarkers such as phosphoproteins and RNAs not present in
original sample thru ex vivo stimulation. The invention provides
fully integrated specimen and information management in a complete
diagnostic cytology laboratory system and controlled conditions
following biopsy, which minimizes variability between tests,
minimizes the risk of biocontamination, and minimizes the effect of
the sample preparation process itself on biomarker expression.
Embodiments of the present invention can be used to facilitate
targeted treatment of the tumors, and optionally also provide a
tissue sample adequacy evaluation such as a cell-count function
and/or other connected analyses.
As illustrated in FIG. 9, traditional cell processing techniques
can use formalin fixation prior to tissue processing and eventually
embed the cells in paraffin. This results in a lot of potential
cellular information being "lost" as shown in FIG. 11. However, the
improved methods described herein and depicted for example in FIGS.
9 and 12 allow for automated processing of live cells, stimulation
of these cells, and then analysis of the cells using the methods
described below.
As one of skill in the art will appreciate, these novel devices,
systems, kits and methods can provide numerous advantages in a
clinical or research setting. For example, they can be used to
provide immediate, near patient, biopsy processing without the need
to send the specimen to a remote laboratory. They can also be used
to standardize and automate biopsy processing in a cost effective
manner. The present invention can provide more detailed molecular
information about the cells than current pathological processes
allow which enables greater sub-classifications of cells in a
biopsy (e.g., cancer cells), optionally using new ex vivo
biomarkers and diagnostic tests. Taken together, the advantages of
the present invention allow for a rapid diagnosis at the point of
care and the subsequent creation of more effective patient specific
treatment regimens.
An example, non-limiting process for using for the devices,
systems, and methods, which are described in more detail to follow,
is shown in the flow chart in FIG. 12. The process can begin by
obtaining a sample of aggregated cells such as the Fine Needle
Aspiration (FNA) step 1201 shown in FIG. 12. The sample is then
disaggregated using the novel techniques described herein and then
dispersed 1203 into isolated chambers. If the sample contains a
mixture of cells of interest and other cells, the sample can be
optionally purified to enrich 1205 the number of cells of interest
in the sample by removing contaminants and cells that are not of
interest. The sample can then be optionally stimulated ex vivo 1207
or otherwise mixed with a test reagent and then aliquots are placed
into new isolated chambers 1209. The aliquots can also be
optionally stimulated ex vivo or otherwise mixed with a test
reagent depending on the assay being performed. The aliquots are
then analyzed for a property of interest. For example, slides can
be prepared from the aliquots for microscopic analysis 1211 or
aliquots can have their cells lysed and the nucleic acids, RNA and
DNA, analyzed, 1213 and 1215, respectively. The results of the
analysis are then communicated to the researcher or clinician who
can take appropriate action, for example, setting a treatment
regimen for a patient from which the FNA was taken. Further, as
illustrated in FIG. 11, the improved methods and devices disclosed
herein can allow a researcher or clinician access to new
information that is "lost" during traditional pathological cell
processing techniques.
I. Devices, Systems, and Kits
A. Devices
In a further embodiment, the invention provides a device or
platform, which is useful, e.g. in the methods of processing and/or
preparing live cells described herein. This device is also referred
to as a cartridge. Some embodiments of the devices of the present
invention are described in more detail below and depicted in FIGS.
1-8.
Such cartridges can contain one or more isolated chambers. An
isolated chamber is any compartment, section, or other utility
holder than can hold a sample of live cells or a sample of fixed,
processed, and/or stabilized cells. For example, the term isolated
chamber includes, but is not limited to, wells, vials, tubes,
slides (e.g., glass), and plates.
The isolated chambers or compartments of the present invention are
suitable for one, some or all of the following functions: (1)
receiving biological specimens via a septum or other sealed
chamber; (2) contained and secured syringe needle storage; (3)
liquid reagent storage available for removal via septum; (4) waste
receipt and storage via septum; (5) sample disruption via liquid
shear and mechanical shear; (6) cell counting and cell
visualization; (7) bead based separations, and (8) containing solid
resins.
Each cartridge can contain one or more isolated chambers depending
on its use. For example, a cartridge can have between 1 and about
200 isolated chambers, between about 1 and 100 isolated chambers,
or between about 1 and 50 isolated chambers. Some embodiments have
about 24, about 48, or about 96 isolated chambers.
In some embodiments, a cartridge has one or more of a first
isolated chamber and one or more of a second isolated chamber. A
cartridge can have a first isolated chamber for holding a sample of
cells. Such a cartridge can also feature one or more second
isolated chambers which hold the dispersed aliquots of the cell
sample. In some embodiments, the second isolated chamber can
contain a predetermined amount of a test reagent in the chamber
before the dispersed aliquot of cells is added. FIG. 2 illustrates
exemplary isolated chambers that may be present for receiving and
handling a sample.
In one embodiment the invention provides a cartridge having
compartments (e.g, isolated chambers) that can be separated from
one another. As illustrated in FIG. 7, the compartments can be
separated and the sample inside may be used for different
analytical tests. For example, one compartment can be sent for DNA
analysis, another for RNA analysis, another for microscopic
analysis, and another for immunohistochemical analysis. For
example, the second isolated chambers on a cartridge can be
separated from the first isolated chamber in some embodiments.
The devices of the present invention can contain a heating element
to maintain the temperature of the cartridge at a desired
temperature, for example, between about 30.degree. C. and
40.degree. C., between about 36.degree. C. and about 38.degree. C.;
or any other desired temperature.
The devices of the present invention can contain a barcode or other
means of indentifying the cartridge and/or the source of the sample
of cells in the device. The barcode or others means of identifying
the cartridge can be used to facilitate specimen identification
and/or patient safety. Such means of identification can interact
with computer based data storage systems, automated cell processing
systems and/or assays, and personal digital assistants. An example
embodiment of a cartridge having a barcode is depicted in FIG.
4.
Some embodiments of the device can include a cell counting
mechanism, as depicted, for example in FIG. 2F. These mechanisms
can be automated systems or manual systems. For example, the cell
counting mechanism can be a hemocytometer or a Cellometer.RTM.
(available from Nexcelom Biosciences, LLC) and potential digital or
optical based counting. As one of skill in the art will appreciate,
other methods of counting cells are well known in the art and can
also be used with the present invention.
The cartridges of any embodiment of the present invention can have
one or more modules or isolated chambers that contain resins,
reagents, solvents, and other materials. A module may be a single
isolated chamber or a set of a plurality of isolated chambers used
for a particular purpose. For example, where multiple manipulations
(e.g. lysing and staining) are involved for a particular test, more
than one isolated chamber may be used as part of that test
"module." For example, one or more isolated chambers can contain
resins with attached nucleic acids, proteins, natural or synthetic
polymers or small molecules. One or more isolated chambers can
contain liquids, e.g. buffers, molecular biology enzymes,
biological molecules including nucleic acid or proteins, or small
molecule chemicals. One or more isolated chambers can contain dry
reagents for on-cartridge solubilizations, e.g. where dry reagents
may include nucleic acid, proteins, natural or synthetic polymers
or small molecules.
In some embodiments, a camera or other digital imaging device is
part of the cartridge or can be used with a cartridge. Accordingly,
the cartridges of any embodiment of the present invention can
include an imaging module that allows cell visualization and
digital image based cell counting (see FIG. 2F) on samples of
volumes between 10 and 500 microliter and said volume is dispensed
into imaging cell by positive displacement or by capillary action.
The imaging device can also be used to take pictures of the sample,
e.g., cellular components within the sample such as protein
localization data.
The cartridges of any embodiment of the present invention can have
a fluidic module that passages cells through micropassages with
diameters of 10 to 500 microns to create wall shear stresses of 100
to 800 dynes/cm2 using volumes between 10 and 1000 microliters.
The cartridges of any embodiment of the present invention can have
an immunodepletion module or isolated chamber. Such an
immunodepletion module or isolated chamber can utilize magnetic
beads or other methods. See, e.g., the magnetic bead products
available from Dynal Biotech, Oslo, Norway. These embodiments are
described in more detail in the methods section.
The cartridges of any embodiment of the present invention can have
one or more modules or isolated chambers that contain or are
designed to contain a biological specimen which can be, for
example, a fine needle aspiration biopsy, core biopsy, biological
fluid sample such as saliva, blood, semen, or vaginal fluid,
harvested tissue from an organism, or cell culture sample. Such a
module can also be referred to as the first isolated chamber and is
depicted, for example, in FIG. 2A and in FIG. 4.
In a further embodiment, the invention provides cartridges, e.g.
suitable for use in the methods and devices described herein, for
example having individual and self-contained modules, the modules
containing media suitable for cell handling and being each sealed
by a septum or other sealing mechanism, said septum or sealing
mechanism being capable of being bypassed or perforation by a tube,
e.g., needle, and resealing upon removal of the tube. In some
embodiments, there are sufficient modules or isolated chambers to
permit unified delivery and removal of all liquid reagents,
biological test specimens, sharps (i.e. needles), etc. into and out
of the cartridge or an analytical device enclosing the cartridge
where the analytical device provides biohazard containment during
analysis. Modules can contain a number of isolated chambers with
associated reagents and devices for performing specific tests or
activities such as cell counting or viability assay. The
embodiments described above are depicted, for example, in FIGS.
1,2, 4, 6, 7, and 8.
As shown in FIG. 5, 501B, the cartridges of the present invention
can have slides stacked in, for example, a staircase configuration
501A that enables the slides to be efficiently utilized to accept
cellular material then subsequently accessed and removed singly by
manual or robotic means. FIG. 5 shows a cross section illustrating
the staircase configuration 501A/B. Thus in a further embodiment,
the invention provides a device which comprises a holder containing
a multiplicity of planar substrates, e.g., glass slides, arranged
in a staircase configuration, wherein the holder restricts lateral
and vertical movement of substrates. Suitable substrates include,
but are not limited to, flat rectangular pieces such as glass
slides, metal plates, or microfluidic devices. The substrates can
have a thickness between 0.1 and 3 mm and/or dimensions of 2-3 cm
wide by 7-9 cm long. As one of skill in the art will recognize,
other substrates or isolated chambers may be arranged in a similar
manner on the cartridges and such embodiments are encompassed
herein.
In some embodiments, the holder on the cartridge has a bottom
portion and a lid portion with each portion containing stair steps
for positioning and preventing movement of a staircase stack of
individual substrates within the holder or relative to the holder.
When stored in the holder, the substrates can have a distal end
where the bottom-most portion protrudes beyond the stack (see FIG.
5 at 501B). This distal end can have one or more printed features,
wells or depressions. These printed features can be labels for
identifying the substrates once they have been used or prior to
use. In some embodiments, the holder has a lid and the distal end
of the substrate is exposed to access through a septum or septa of
the lid. The holder can be mounted and dismounted into a frame, for
example, a frame with an SBS-compatible footprint. Optionally, the
holder can have a small hole or plurality of holes in the
bottom.
FIG. 5 illustrates a top view of an exemplary holder. The stack of
slides can be addressable from above according to standardized row
and column spacing making the system ideal for automated or manual
use. The holder can have a lid with septa above each substrate for
addressing positions above each substrate independently. For
example, the holder can have an O-ring or gasket sealable chamber
above each addressable position of each substrate. These O-ring or
gasket sealable chambers can be of any shape desired, for example,
circular or rectangular or square.
Positioning the holder and substrates in a predetermined manner can
allow multiple delivery of fluid sample of volumes ranging from 1
nanoliter to up to 0.5 milliliter to the substrate through the use
of a pipette, syringe needle, or pintool. The holder, in some
embodiments, restricts lateral or vertical displacement of
substrate during fluid delivery and restricts movement of substrate
during fluid delivery. In other embodiments, the holder permits
vertical rotation but restricts lateral movement of substrates.
This flexibility can allow automated or manual multiple fluid
delivery to and removal from each isolated chamber using a wide
array of methods and systems.
In some embodiments, the holder can be disassembled with the lid
portion removed and the bottom portion can be mounted on a stand
that positions a protrusion through the hole in the bottom portion
of the holder. The substrates can be uniquely presented while on
the stand protrusion for sequential gripping by human hand or a
robotic grip tool. This allows for sequential removal of
substrates, e.g., bottom slide first and top slide last, to prevent
scraping of substrates over deposited samples on each sample
thereby allowing stable transportation of slide-based cellular
material. Example disassembled views of cartridges having
embodiments of the above described holder are disclosed in FIGS. 6
and 8.
The fluidic samples can be delivered to the substrates in an
automated system or manually. In some embodiments, the fluidic
samples are sequentially delivered to the substrates in a
predetermined order. Suitable fluidic samples include, but are not
limited to, solutions, emulsions, suspensions, or
polymer-containing mixtures. For example, the fluidic sample can
be, but is not limited to, biological or chemical materials, e.g.,
small organic or inorganic molecules, proteins, nucleic acid,
cells, particles, volatile and non-volatile solvents, polymers, or
fixatives.
In a further embodiment, the invention further provides well plates
useful in the cartridges of the invention. Currently, there is a
need for a well plate technology that allows components (open
tubes, sealed tubes, syringes, pipette tips, etc) to be locked in
position so that they will not fall out of position if the plate is
held sideways or upside down. Furthermore, well plate technology
allows the locked-in component to be removed by a standard
(Cartesian) pipette tool, utilized, and then returned to a
locked-in position, without requiring even a tip ejector on the
pipette tool. In some embodiments, the present invention is
directed to a well plate comprising a planar surface with a
multiplicity of wells that is manufactured such that two adjacent
positions (called "dual well") on the well plate are connected by
an intervening space allowing lateral transit of a component.
The component can be an open tube, sealed tube, tube with septums,
transparent tubes, any container, syringe, needle, blade, or
pipette tip. In some embodiments, the component contains a rigid
piece of material of square, circular, or other shape of suitable
thickness to pass through a locking mechanism and conforms to the
shape of the locking mechanism of a lock-in well.
In some embodiments of the dual-well plate, the first adjacent
position of the dual well (called a "lock-in well") contains small
tabs that allow components to be locked in position and resist
movement, especially withdrawal of the component in the vertical
direction when the plate is facing upward. These dual-well plates
can also have a lock-in well containing a compliant gasket that is
partly compressed when a component is locked into the lock-in well
and this gasket prevents movement of the locked-in component.
In some embodiments, the component contains a "fitting" addressable
by a tool (e.g., a pipettor or syringe) wherein the fitting (e.g.,
a luer lock) allows a snug and airtight fit with the tool. The tool
can mount the component that is locked into the lock-in well and
push down on the component to further compress the gasket thereby
allowing the component to reside in a position that allows lateral
transit within the intervening space of the dual well from the
first position (lock-in well) to the second position of the dual
well, called the "release well." In some embodiments, the component
can freely travel out of and into the release well when moved
vertically by the tool. The tool can also deliver a component fully
into the lock-in well and, when the well plate is anchored to a
surface, the tool can move vertically away from the wellplate,
thereby dismounting the component while the component is locked in
the lock-in well.
The tool and the plate can each be mounted on a computer controlled
gantry system allowing movement of the tool and/or plate in
Cartesian coordinates (x, y, z) thereby allowing the tool to
deliver components to the lock-in well and then leave components in
the lock-in well, mount components in the lock-in well and move
them to the release well, remove components from release well and
free them from the well plate. Such a setup can also allow the tool
to deliver components to the release well and leave components in
the release well by means of a tip ejector.
Some computer-controlled systems using the tool and plate can be
set up to not require the use of angular motion (theta-axis)
thereby preventing positional rotation reorientation of the
component with respect to the well plate while the component is in
the well plate or removed from the well plate.
In some embodiments, the dual-wells can be used to allow fluid to
flow through a predetermined path that has been designed to remove
contaminants from the sample. Such a dual well system is
illustrated, for example, in FIG. 2 at wells C and D.
B. Systems
The present invention is also directed to systems that utilize the
cartridges and methods described herein. For example, a system can
include one or more of the inventive cartridges, either
individually or as part of a kit, and an analytical device (also
referred to herein as an apparatus) capable of interacting with the
cartridge to obtain at least one analytical result. For example,
the system can be used at the point of care to obtain a medical
diagnosis for a patient.
Accordingly, some embodiments of the invention are directed to an
analytical device for performing operations on a cartridge
containing individual modules (e.g, isolated compartments) wherein
the device comprises one or more holders for one or more syringes,
the syringes having hollow needles and being oriented above a
platform on which the cartridge is located such that the needles
can be directed to individual modules of the cartridge using a
Cartesian coordinate system (x, y, z) by computer controlled motion
control to apply suction and dispense contents to the modules of
the cartridge, the modules each having a septum which can be
penetrated by the needles, but which otherwise isolates the
contents of the modules from the environment. Examples of some
embodiments of these cartridges are depicted in FIGS. 1, 4, and 8.
In some embodiments the holder can pick up a syringe or tool
provided by the cartridge.
The analytical device can have a means for receiving a cartridge of
the present invention. For example, a slot, opening, hole, or other
area capable of receiving a cartridge. Once a cartridge has been
placed in the means for receiving, in some embodiments, a door
closes to seal the means for receiving and thereby fully enclosing
the cartridge within the analytical device. This door closure can
be manually performed or automatic. These fully enclosed versions
can be especially useful in preventing potential biohazards from
being spilled or otherwise released into a lab or clinical
environment.
In some embodiments, the systems for using the analytical device
and cartridge do not require or use vacuum utility; air service
utility; natural gas utility; and can be free of all reagents lines
(process fluid, analytical reagents, and waste streams) and
attached bottles. That is, the device is fully self contained.
The cartridges used in such a system, or used independently, can
also be self-contained. This means the cartridge comes pre-loaded
with all necessary tools (e.g., pipette tips) and reagents (e.g., a
test reagent, dye, or other compound) required to perform a desired
assay either manually or using an automated system. For example,
the cartridge could be pre-loaded with a panel of cancer
therapeutics (e.g., one each in an isolated chamber such as a well
in a plate) and all the necessary tools for disaggregating and
dispersing a sample into these wells.
The systems of the invention can include an internal imaging
capability to address a module of a cartridge. This system can be
used to generate data which is then outputted to a user or other
device.
The analytical device can also include components necessary for
thermal incubation to preserve cell viability while tests are run
on the sample. The incubation portion of such an analytical device
can hold one or more cartridges, optionally in a predetermined
order (e.g., chronological order based on time of sample
extraction).
Any of the foregoing devices that can also attach sample
identifiers to modules of the cartridge and transmit sample
information and process information via communication lines to
other devices or can display a result for a user to examine.
C. Kits
The present invention is also directed to kits containing a device
of the present invention. Example kits include one or more
cartridges described herein packaged in a container. The kits can
further include printed instructions for use, reagents and buffers,
molecular probes, one or more test reagent as discussed below,
disaggregation or distribution tools such as pipetters or needles,
and other items useful in performing the methods described
below.
In some embodiments, these kits can be sterilized using methods
known in the art and packaged in a manner to preserve the
sterilization. The kits can be sold as individual kits or in a
multipacks. The kits can also be designed in a manner such that
they are tamper resistant or designed to indicate if tampering has
occurred.
A kit can include a cartridge for analysis as described herein that
can be used manually rather than through the use of an automated
apparatus. In such cases, reagents and equipment required to
conduct the test or purpose of the cartridge can be provided as
part of the kit. The equipment, e.g. syringes needles, pipettes,
etc., may be preloaded onto the cartridge or may be outside of the
cartridge and provided as part of the kit. In other embodiments,
the equipment may be supplied by the user and not as part of the
kit. Similarly, test reagents that are used with the kit may be
preloaded into particular isolated chambers of the cartridge or
packaged outside the cartridge for application by the used. Kits
according to these embodiments can be packaged as, for example, a
single cartridge for a single test, a single cartridge loaded or
prepared for multiple tests, or multiple cartridges for multiple
tests.
Optionally, the kit also contains directions for properly using the
cartridge and other necessary items, e.g., reagents, as part of an
assay or method such as those described herein. For example, the
kit can contain a notice or printed instructions. Such printed
instructions can be in a form prescribed by a governmental agency
regulating the manufacture, use, or sale of pharmaceuticals or
biological products, which notice reflects approval by the agency
of the manufacture, use, or sale for human administration to
diagnose or treat a condition that could be treated using
information derived from the assays, methods, and devices described
herein. In some embodiments, the kit further comprises printed
matter, which, e.g., provides information on the use of the kit to
process cells or a pre-recorded media device which, e.g., provides
information on the use of the kit to process cells.
"Printed matter" can be, for example, one of a book, booklet,
brochure or leaflet. The printed matter can describe experimental
assays and/or protocols for processing cells according to the
present methods. Possible formats include, but are not limited to,
step-wise instructions, a bullet point list, a list of frequently
asked questions (FAQ) or a chart. Additionally, the information to
be imparted can be illustrated in non-textual terms using pictures,
graphics, or other symbols.
"Pre-recorded media device" can be, for example, a visual media
device, such as a videotape cassette, a DVD (digital video disk),
filmstrip, 35 mm movie, or any other visual media device.
Alternately, pre-recorded media device can be an interactive
software application, such as a CD-ROM (compact disk-read only
memory) or floppy disk. Alternately, pre-recorded media device can
be, for example, an audio media device, such as a record,
audiocassette, or audio compact disk. The information contained on
the pre-recorded media device can describe experimental assays
and/or protocols for processing cells according to the present
methods.
II. Methods
The present invention is directed to novel methods of processing
cellular samples, in particular aggregated cells or solid tumors
which can be used in a clinical or research context. These methods
include disaggregating and dispersing an aqueous solution
containing live cancer cells obtained from a subject into at least
one test aliquot in a first isolated chamber; optionally purifying
or manipulating the sample to increase the percentage of target
cells relative to other contaminating cell types by removing the
contaminating cells; distributing the purified live cancer cells
into one or more second isolated chambers for analysis,
manipulation, or stimulation; and stabilizing the distributed live
cells to permit cellular and/or molecular analysis of the
distributed cells. In some embodiments, the stabilized and
distributed cells can be live cells or dead cells, depending on the
desired outcome and the cellular assay of interest.
Other methods of the present invention include methods for
processing or preparing cancer cells from a solid tumor comprising:
a. disaggregating and dispersing live cancer cells obtained from a
solid tumor into at least one test aliquot in at least one first
isolated chamber; optionally purifying or manipulating the live
cancer cells to remove contaminants; distributing the purified
live, purified cancer cells into one or more second isolated
chambers for analysis; and stabilizing the distributed cells to
permit cellular and/or molecular analysis of the cells.
The methods of the present invention allow live cells to be
processed rapidly. The cells can be processed in a live state with
minimal cellular activation or stress (e.g., environmental stress,
temperature induced stress, metabolic stress, or chemically induced
stress). The term "minimal cellular activation or stress" is
defined by minimal changes in background noise of cell signaling
and cell stress pathways compared to stimulation. For example, as
seen in FIG. 17, a comparison of FOS (or c-fos) induction in cells
before and after disaggregation and/or dispersion can show minimal
induction (3-5 fold) of FOS or other early response genes or
biological stress indicators compared to stimulated samples (20-30
fold increase).
Another advantage of the methods of the present invention is that
they can use very low numbers of cells in the original sample. For
example, in some embodiments the total number of aggregated cells
or solid tumor cells processed is between about 1.times.10.sup.3
and 1.times.10.sup.7.
The methods of the present invention also allow for rapid sample
processing. In some embodiments, the stabilization of the
distributed live cells is completed within about one hour, about
two hours, about three hours, or about four hours of obtaining the
sample from the subject. This surprisingly short processing time
eliminates the need for cell culturing while maintaining high rates
of cell viability, for example, over about 50%, 60%, 70%, 75%, 80%,
85%, 90%, or 95%. In some embodiments, the rate of cell viability
is about 70% or about 75%. The term "about" when used in
conjunction with a number, for example a percentage, means plus or
minus 10% of the number. For example, the term "about 60%" includes
between 54% to 66%.
A. Obtaining the Sample
The sample used in the methods and devices described herein can be
obtained in a variety of ways. The sample can be live cells taken
from a subject, such as a mammal (e.g. a human) or another living
organism. For example, the sample can be a biopsy taken from a
human patient in a clinical setting for analysis which is
eventually used to help determine the proper clinical diagnosis and
course of treatment.
The sample in some embodiments can also be any group of cells or
single cell, aggregated or disaggregated, that is of interest in a
research or clinical setting. For example, solid tumors as well as
individual cells such as lymphomas or cells that have been
disaggregated using other means than described herein, such as, by
using trypsin. These samples can be from existing cell lines,
xenografts, or patient specimens that are examined for reasons
other than to provide a clinical diagnosis. Such samples can be
analyzed to further characterize the cells and their responses to
specific test reagents. These applications can be useful as part of
drug development and screening assays to identify new compounds or
improve the administration of existing compounds.
In some embodiments, the methods described herein can be used with
any type of aggregated cells or tumor cells. For example, they can
test and process carcinomas or sarcomas. Example cancers that can
be tested with the present methods include, but are not limited to,
colon cancer, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, cervical cancer, lung
cancer, small cell lung carcinoma, kidney cancer, liver cancer,
brain cancer, skin cancer, and bladder cancer. These cancers can be
from a human, other mammal, or a xenograft of human cancer cells
removed from a non-human mammal (e.g., a mouse).
In some embodiments, the tissue sample is a portion of a solid
tumor or a complete tumor. Such a tissue sample containing tumor
cells for use in the present invention may be obtained by any
method as is know in the art, for example, by taking a biopsy from
a patient. Suitable biopsies that may be employed in the present
invention include, but are not limited to, incisional biopsies,
core biopsies, punch biopsies and fine needle aspiration biopsies,
as well as excisional biopsies. In some embodiments, the biopsy is
obtained by fine needle aspiration (FNA) of a tumor.
Fine Needle Aspiration (FNA) biopsy is performed with a fine needle
sometimes attached to a syringe and other times used independently.
Aspiration biopsy or FNA may be employed in the present invention
to obtain a cancer sample. FNA biopsy may be a percutaneous
(through the skin) biopsy or alternatively through the lumen of an
organ such as the bronchus, esophagus, stomach, or intestine. FNA
biopsy is typically accomplished with a fine gauge needle (21 gauge
or finer, e.g., 22 gauge or 25 gauge). The area is first cleansed
and then usually numbed with a local anesthetic. The needle is
placed into the region of organ or tissue of interest. Once the
needle is placed a vacuum may be created with the syringe, or
alternatively capillary action within the needle alone may be
utilized, and multiple in and out needle motions are performed. The
cells to be sampled are brought into the lumen of the needle and
sometimes the hub of the needle through a micro-coring action of
the bevel of the needle as it passes through the tissue. Three to
six separate samples are usually made. Metastatic cancer sites such
as lymph nodes and liver are good candidates for FNA biopsies. FNA
procedures are typically done using ultrasound or computed
tomography (CT) imaging.
A core needle biopsy (or core biopsy) is performed by inserting a
small hollow needle through the skin and into the organ. The needle
is then advanced within the cell layers to remove a sample or core.
The needle may be designed with a cutting tip to help remove the
sample of tissue. Core biopsy is often performed with the use of
spring loaded gun to help remove the tissue sample. Core biopsy is
typically performed under image guidance such as CT imaging,
ultrasound or mammography. The needle is either placed by hand or
with the assistance of a sampling device. Multiple insertions are
often made to obtain sufficient tissue, and multiple samples are
taken. Core biopsy is sometimes suction assisted with a vacuum
device (vacuum assisted biopsy). This method enables the removal of
multiple samples with only one needle insertion. Unlike core
biopsy, the vacuum assisted biopsy probe is inserted just once into
the tissue through a tiny skin nick. Multiple samples are then
taken using a rotation of the sampling needle aperture (opening)
and with the assistance of suction. Thus, core needle biopsy or
vacuum assisted needle biopsy may be employed in the present
invention to obtain a tissue sample.
Endoscopic biopsy is a common type of biopsy that may be employed
in the present invention to obtain a sample. Endoscopic biopsy is
done through an endoscope (a fiber optic cable for viewing inside
the body) which is inserted into the body along with sampling
instruments. The endoscope allows for direct visualization of an
area on the lining of the organ of interest; and collection or
pinching off of tiny bits of tissue with forceps attached to a long
cable that runs inside the endoscope of the sample. Endoscopic
biopsy may be performed on, for example, the gastrointestinal tract
(alimentary tract endoscopy), urinary bladder (cystoscopy),
abdominal cavity (laparoscopy), joint cavity (arthroscopy),
mid-portion of the chest (mediastinoscopy), or trachea and
bronchial system (laryngoscopy and bronchoscopy), either through a
natural body orifice or a small surgical incision. Endoscopic
ultrasound-guided fine needle aspiration biopsy may also be
performed on lung or mediastinal lymph nodes, pancreas, or liver
using a trans-esophageal, trans-gastric or trans-duodenal
approach.
Surface biopsy may be employed in the present invention to obtain a
cancer sample. This technique involves sampling or scraping of the
surface of a tissue or organ to remove cells. Surface biopsy is
often performed to remove a small piece of skin.
B. Cell Dispersion and Disaggregation
The sample obtained for processing can be prepared for analysis by
separating the cells from one another (if aggregated) and then
dispersing the separated cells into test aliquots within the
cartridges described herein or into another suitable container.
This process may consist of multiple steps including dispersion and
counting/viability assays.
Compared to samples from surgically-excised tumors, the samples
used in the methods of the present invention can contain relatively
small numbers of cells (e.g., tumor cells), which, in the absence
of the methods disclosed herein, can in some cases limit
utilization of these small samples in many current molecular
diagnostic technologies. Further, FNA samples from solid tumors or
solid tumor cells obtained using other methods can contain
extremely large clumps of cells (>500 cells), which prohibit
uniform distribution of the specimen into multiple testing
compartments.
Disaggregating and dispersing such cells while not killing or
unduly activating stress response pathways within the cells is a
delicate process that requires precise methods and techniques. As
used herein, disaggregation means separating cells or providing an
approximately homogeneous sample of cells in such a way that a
sample containing the cells is capable of being dispersed into
multiple relatively uniform samples. For example, in
anchorage-dependent cells such as endothelium, sustained
unidirectional laminar shear forces at arterial levels (10 to 25
dyne/cm.sup.2) can cause rapid changes in metabolism (prostacyclin
and NO production) as well as rapid changes in gene expression with
FOS mRNA and FOS protein enhanced in less than an hour. Several
other cell lines such as CHO and HELA also display FOS induction
after sustained exposure to unidirectional shear stress. These
studies typically deploy flow chambers where cells are exposed to
shear stresses for minutes to hours to days to alter phenotype.
See, e.g., Diamond S L, Eskin S G, McIntire L V. Fluid flow
stimulates tissue plasminogen activator secretion by cultured human
endothelial cells. Science. 1989 Mar. 17; 243(4897):1483-5.
Distinct from fluid shear studies, mechanical perturbation
(substrate stretching or induced deformation) can activate
stretch-activated ion channels and can cause calcium mobilization.
Turbulent shear stresses are typically more detrimental than
laminar shear stresses in that interactions with collapsing films
of bursting oxygen bubbles are particularly cytolytic. Additives
such as pluronic F68 or bovine serum are cytoprotective, but partly
act via surfactant effects that prevent cells from associating with
air bubbles.
The present inventors have surprisingly found that using
predetermined amounts of laminar fluid shear stress can effectively
disrupt aggregates of cells without killing the cells and
triggering only a minimal stress response or no stress response in
the cells. This disaggregation can be done, for example, by drawing
the cells into a needle or tube of a predetermined size and
ejecting the cells, e.g. into a well, and repeating as necessary.
The exposure time to laminar shear stress is minimized to reduce
shear activation of cells during fluid mechanical disruptions of
the specimens.
The proper conditions for disaggregation of a sample can be
calculated to identify the equipment and protocol needed. The
aggregation state at any instant of a cellular system is defined by
its population size distribution. A system may be monodisperse
(e.g. all singlets or aggregates containing small numbers of cells,
for example all 20-mers or less) or polydisperse with aggregates
ranging between singlets and a range of k-mers, where k is a large
number greater than, for example, 20. In cell culture lines,
aggregates are homotypic. However, FNAs are heterotypic in that
they contain multiple cell types. The mathematics of aggregation
and fragmentation processes that evolve in time are well developed.
Depending on the complexity, population balance equations can be
solved analytically (simple homotypic aggregation), numerically
(complex homotypic aggregation), or by Monte Carlo simulation
(heterotypic aggregation/fragmentation). For FNA disruption, the
fundamental process is dictated by the fragmentation kernel F which
depends on prevailing flow fields, aggregate size, and buffer
conditions. For a population of sizes undergoing fragmentation
(single component with each particle breaking into two smaller
particles), the fragmentation balance may be expressed as:
d.function.d.function..times..times..times..infin..times..times..function-
. ##EQU00001## where c.sub.k(t) is the concentration of k-sized
particles (or k-mers) at time t and a.sub.k is the net breakup rate
of k-mers and b.sub.i|k is the average number of i-mers produced
upon breakup of a k-mer. Thus, F.sub.ij=a.sub.i+jb.sub.i|i+j/2
gives the net rate that (i+j)-mers break into i-mers and j-mers.
The Fragmentation Kernal F is spatially dependent in tube flow
(high near the wall, zero in the center) and is also dependent on
the ratio of the aggregate size to the tube diameter. In the
manipulations after biopsy, the FNAs will be highly dilute (cell
volume/sample volume<<1) so that suspension dynamics
involving radial migration to the walls of the smallest particles
are not important. Fragmentation of an aggregate can range from
binary fissure to pure ginding (loss of singlets from the
aggregate). Fragmentation Kernals are not known for tumor
aggregates in FNAs. As one of skill in the art will appreciate,
empirical fragmentation rates for clusters in shear flow are
power-law relationships based on the average shear rate G.sub.avg
and the aggregate hydrodynamic radius or collision radius
R.sub.hyd. For example, a common form is:
a.sub.i=A*(G.sub.avg).sup.y(R.sub.hyd).sup..gamma. with A and y
determined experimentally and .gamma.=2. For a tumor aggregate of
i-cells where each cell has a radius R.sub.o, then
R.sub.hyd=R.sub.o, (i).sup.1/Df where D.sub.f is the fractal
dimension (D.sub.f.about.1.7 to 2.5).
FNAs, and other cell aggregates, can be complex objects with
multiple cell types and various matrix constituents. In considering
the disruption of FNAs, the fragmentation of the large tissue
samples derived from the patient into a subpopulation is primarily
an issue of disruption of junctions between tumor cells and
secondarily disrupting integrin-dependent adhesion between the
tumor cell and the underlying matrix.
Shear induced disaggregation of biopsy samples, e.g., FNAs, in
tubes: One method of disaggregating cells is through the use of
shear stress. As mentioned above, laminar shear stress is
preferred. Laminar shear stress can be generated in tubes.
For laminar shear flow in a tube (Reynolds number<2100), the
shear stresses are greatest near the tube wall and are zero in the
center of the tube where fluid is simply translating downstream.
Wall shear stress t.sub.w and transit time t.sub.transit may be
defined as: t.sub.w (dyne/cm.sup.2)=4 mQ/(.pi.R.sup.3) and
t.sub.transit=(LA)/Q for volumetric flow rate Q though a tube of
cross-sectional area A=.pi.R.sup.2 where: Q=v.sub.avgA for
Q[=]cm.sup.3/s, v.sub.avg[=]cm/s, and A[=]cm.sup.2. The average
transit time across a length of tubing L is defined from
v.sub.avg=L/t.sub.transit such that t.sub.transit=L/v.sub.avg=L
A/Q. The viscosity of water is 0.01 Poise at room temperature.
Additives such as glycerol, pluronic F68, dextran, polyethylene
glycol (PEG) can all enhance the viscosity of the fluid phase. At
constant flow rate and geometry, increasing the viscosity will
increase the shear forces. Entrance length effects are fairly
minimal in small diameter tubes. For a commonly used length of 1''
syringe and syringe gauges (G) and water perfusion buffer (1 cP),
wall shear stresses (dpc, dyne/cm.sup.2) and transit times are
given in Table 1.
TABLE-US-00001 TABLE 1 Relationship of needle gauge, wall shear
stress (dpc, dynes/cm.sup.2), and transit time (msec) for 1''
needle perfused with water buffer at 1 mL/s (viscosity = 1 cP).
Transit Time Onsec) Needle ID Radius Area Shear Stress (dpic) Avg.
Velocity (mils} 1-in. syringe Gauge inches cm cm.sup.2 Q = 1.0 mL/s
Q = 1.0 mL/s (Q = 1.0 mL/s) 10 0.109 1.346E-01 5.693E-02 5.219
17.58 144.6115 11 0.094 1.194E-01 4.477E-02 7.484 22.34 113.7226 12
0.085 1.080E-01 3.661E-02 10.121 27.32 92.9884 13 0.071 9.017E-02
2.554E-02 17.367 39.15 64.8795 14 0.063 8.001E-02 2.011E-02 24.859
49.72 51.0825 15 0.054 6.858E-02 1.478E-02 39.475 67.86 37.5300 18
0.047 5.969E-02 1.119E-02 59.870 89.34 28.4306 17 0.042 5.334E-02
8.938E-03 83.898 111.88 22.7033 18 0.033 4.191E-02 5.518E-03
172.965 181.22 14.0158 19 0.027 3.429E-02 3.694E-03 315.797 270.72
9.3825 20 0.023 2.921E-02 2.680E-03 510.876 373.07 6.8084 21 0.0195
2.477E-02 1.927E-03 839.292 519.01 4.940 22 0.0155 1.969E-02
1.217E-03 1669.184 821.45 3.0921 23 0.0125 1.588E-02 7.917E-04
3182.506 1263.06 2.0110 24 0.0115 1.461E-02 6.701E-04 4087.011
1492.27 1.7021 25 0.0095 1.207E-02 4.573E-04 7249.841 2186.73
1.1616 28 0.0095 1.207E-02 4.573E-04 7249.841 2186.73 1.1616 27
0.0075 9.525E-03 2.850E-04 14733.826 35138.49 0.7240 28 0.0065
8.255E-03 2.141E-04 22633.893 4671.07 0.5438 29 0.0065 8.255E-03
2.141E-04 22633.893 4671.07 0.5438 30 0.0055 6.985E-03 1.533E-04
37360.377 6524.05 0.3893 31 0.0045 5.715E-03 1.026E-04 68212.157
9745.80 0.2606 32 0.0035 4.445E-93 6.207E-05 144975.692 16110.41
0.1577 33 0.0035 4.445E-03 6.207E-05 144975.692 16110.41 0.1577
Laminar tube flow is one example of moderate extensional flow.
Impinging flows such as a tube directed at a nearby flat surface
are highly extensional. The cellular suspension experiences the
extensional forces for fleeting periods of time at the exit of the
tube before entering a low shear environment. The magnitude of the
extensional forces are easily controlled by tube diameter, flow
rate, and distances from the flat surface. Routine motion control
and micromanipulation can control distances with an accuracy within
10 microns. Also, entrance of fluid into a needle or exit of fluid
from a needle can create strong elongational flows. By use of
varying lengths and inner diameters (Gauge), it is possible to
distinguish disaggregation due to wall shear stress exposure from
that cause by entrance or exit into the needle.
Based on the calculated force and equipment needed, the cells of
the tissue sample can be passed through a tube having a diameter of
10 to 500 microns using volumes between 10 and 2000 microliters to
create wall shear stresses of 100-800 dyne/cm.sup.2. In some
embodiments, the cells are passed through a 22 gauge or 18 gauge
needle. In some embodiments, the cells are exposed to laminar wall
shear stresses of about 100 to about 800 dyne/cm.sup.2, laminar
wall shear stresses of about 300-about 500 dyne/cm.sup.2; or
laminar wall shear stresses of about 350-about 450 dyne/cm.sup.2.
The cells can be exposed to the laminar wall shear stress for
between about 10 msec to about 500 msec or longer, depending on the
amount of force needed and the type of cell.
In some embodiments, the viscosity of the media is adjusted, in
order to provide the proper shear force.
The disaggregation step can be repeated as necessary until a
suitable sample for analysis has been produced. In some
embodiments, at least about 70%, about 80%, about 90%, or more than
about 90% of the cells are dispersed into clumps of 1-100 cells.
The clumps can also be groups of 5-100 cells, 10-100 cells, 10-25
cells, or 5-25 cells. Preferably the clumps have fewer than 15
cells, for example, 1-10 cells per clump.
The disaggregation step can also involve adding a compound to aid
in disaggregation or to prevent activation of a stress response in
the cells. For example, any of the following can be added to the
cells during the disaggregation step a physiologically acceptable
antioxidant; a mucolytic agent; an agent capable of reducing
disulfide bonds, e.g., N-acetyl-L-cysteine or dithiothreitol; a
physiologically acceptable chelating agent, e.g., EDTA; and/or one
or more membrane-protecting surface active agent such as a nonionic
surfactant, e.g. polyethylene glycol, a polyethoxylated fatty acid,
or an ethylenoxide and propylenoxide block copolymer, for example
Pluronic F-68 (BASF).
The dispersion process can also involve suspending the cells or
clumps of cells in a serum-free isotonic saline solution, e.g.,
about 0.9% w/v sodium chloride in sterile water, optionally further
comprising physiologically acceptable buffers and salts; e.g., a
saline solution selected from lactated Ringer's solution, acetated
Ringer's solution, phosphate buffered saline, TRIS-buffered saline,
Hank's balanced salt solution, Earle's balanced salt solution,
standard saline citrate, HEPES-buffered saline.
As one of skill in the art will appreciate, the cellular
disaggregation process can be done manually, for example by a
person using a pipetter or a needle, or by using an automated
process such as an air or fluid driven automated fluidic processing
device. Both manual and automated disaggregation processes are
encompassed by the various embodiments of the present
invention.
In some embodiments, the tissue sample is disaggregated and
dispersed while response pathways, including but not limited to
cellular signal transduction and stress response pathways, are not
activated in comparison to ligand stimulation, for example wherein
the dispersion does not activate FOS expression in comparison to
EGF stimulation.
C. Sample Purification/Enrichment
Aggregated cells can be composed of multiple cell types and often
only very few or one of those cell types is the target for
examination and analysis. In these cases, the cells can be
disaggregated into smaller clumps and/or individual cells and then
the mixture is purified to remove contaminants, including cells
that are not of interest, to purify the sample and enrich it by
providing a higher percentage of the cells of interest than in the
original mixed cellular sample. As used herein, "purify" or
"enrich" means to increase the ratio of the number of target cells
(e.g., tumor cells or other cell being analyzed) to the number of
non-target cells or parts of cells that might otherwise interfere
with analysis.
For example, biopsy specimens from solid tumors are composed of a
mixture of cells including both the cells of interest (e.g. tumor
cells) and contaminating normal cellular elements (hematopoietic
cells, hepatocytes, vasculature, etc). In an exemplary embodiment,
the contaminating elements are removed using antibodies specific to
the contaminating elements or by using antibodies specific to the
target cells, depending on the protocol. The antibodies may be
bound to a substrate, for example, a plastic surface, e.g., the
wall of a plate, or plastic or plastic-coated beads, e.g., magnetic
beads, either directly, or through a second antibody recognizing
the first antibody, so as to remove the contaminating materials
from the cells of interest.
D. Distributing the Cells
Once the cells have been disaggregated and optionally purified
(only if necessary), the sample of cells is distributed into one or
more isolated chambers (e.g., one or more of the second isolated
chambers discussed above) within a cartridge of the present
invention or another suitable container.
The cells of interest may if desired be further dispersed using
techniques as described in B above, and then they are distributed
into aliquots for exposure to test reagents and/or other analysis
and testing. In some embodiments, the aliquots are distributed
among some or all of the wells in a customized cartridge. The
aliquots are exposed to desired test reagents, for example to one
or more ligands to stimulate cell proliferation, and signal
transduction.
The aliquot distributed to the second isolated chamber or other
substrate for testing can vary depending on the number of cells
needed and other experimental conditions known to one of skill in
the art. In some embodiments, the test aliquot has a volume of less
than 2 mL, less than 200 .mu.L, or between 1 .mu.l and 200 .mu.L.
As one of skill in the art will appreciate, the volume can be
varied outside these example ranges if needed so long as a suitable
number of cells are available in the suspension for testing.
In some embodiments, at least one isolated chamber that has
received a test aliquot is designated as a control. This control
can be reassayed for viability and level of stress, undergo
cellular counting processes, or receive additional reagents or
control substances to provide a positive or negative control for
data analysis of the other chambers.
The distributed cells can be suspended in any medium that is
suitable for the cell type. For example, the test aliquots of
distributed cells can be in a serum-free minimal nutrient medium.
The serum-free minimal nutrient medium can have essential amino
acids, salts (e.g., potassium chloride, magnesium sulfate, sodium
chloride, and sodium dihydrogen phosphate), glucose and vitamins
(e.g. folic acid, nicotinamide, riboflavin, B-12); and any other
component necessary for proper processing or analysis of the cells.
Suitable serum-free nutrient mediums include Dulbecco/Vogt modified
Eagle's minimal essential medium (DMEM) or RPMI.
In some embodiments, the test aliquots are distributed into wells
in a plastic plate, e.g., a 96 well plate, wherein the walls of the
wells are coated with a physiologically acceptable hydrogel or oil,
e.g., polyethylene glycol, dextran, alginate, or silicone.
E. Test Reagents and Testing
The test aliquots can be exposed to a variety of test reagents
either in the cartridge or after separating one or more of the
isolated chambers form the cartridge. An advantage of the methods
and devices herein is that the test reagent can be added at the
point of care and/or can come preloaded in specified wells of the
cartridge. This allows the testing of ex vivo biomarkers,
optionally at the point of care, using live cells. These methods
and devices can be used with specific test reagents to manipulate
samples ex vivo to facilitate the development of novel predictive
biomarkers, monitor and determine cellular sensitivity to specific
pharmaceutical agents, and other uses that one of skill in the art
will appreciate.
For example, a sample of a solid tumor from a patient can be
disaggregated, distributed, and then tested against a panel of
currently available cancer therapeutics at the point of care. The
samples can then be stabilized and/or fixed if necessary and
analyzed. Depending on the results for each test reagent, the
physician can quickly determine which therapeutics will be most
effective on the individual patient's tumor at the point of care.
This personalized medicine provides numerous benefits, in
particular, the use of targeted cancer therapeutics and regimens in
a rapid, cost effective manner.
Embodiments of the invention are directed to analyzing the
distributed cells (e.g., cancer cells) by administering at least
one agent to produce a measurable quantitative or qualitative
effect on a target ex vivo biomarker or biomolecule. The
quantitative or qualitative effect can be the activation or
inhibition of a cellular pathway. Exemplary cellular pathways
include, but are not limited to, a metabolic pathway, a replication
pathway, a cellular signaling pathway, an oncogenic signaling
pathway, an apoptotic pathway, and a pro-angiogenic pathway. For
example, the quantitative or qualitative effect can be a
measurement of an agonistic or antagonistic effect on a G-protein
coupled receptor or a receptor tyrosine kinase, such as, epidermal
growth factor receptor (EGFR) and the downstream pathways.
The quantitative or qualitative effect measured can be the
expression level of a gene, such as, an immediate or delayed early
gene family member. Suitable immediate or delayed early gene family
members include, but are not limited to, FOS, JUN and DUSP
1-28.
The effects of the presence or absence of a test reagent can also
be determined by detecting an ex vivo biomarker, for example, a
post-translationally modified protein, ions, or enzymes.
Suitable test reagents can include, but are not limited to, one or
more of the following: a pharmaceutical agent, a chemical compound,
an agent for stimulating a cell, a polypeptide, a polynucleotide,
an antibody, an Fab fragment, an Fc fragment, RNA, miRNA, siRNA and
a phosphoprotein. As discussed above, the administration of a
reagent can be followed by measuring a quantitative or qualitative
effect on a target ex vivo biomarker or biomolecule of the
dispersed or distributed cell.
For certain analytical methods, the test reagent can be a
detectable agent. The detectable agent can be used individually or
as conjugated or otherwise connected to another compound (e.g., a
detectable agent conjugated to an antibody). Suitable detectable
agents include, but are not limited to, an enzyme, fluorescent
material, luminescent material, bioluminescent material,
radioactive material, positron emitting metal using a positron
emission tomography, or a nonradioactive paramagnetic metal
ion.
Other suitable test reagents include tumor-cell stimulatory
ligands, such as, a growth factor (e.g. EGF, insulin, VEGF), or a
hormone, e.g., estrogen or an estrogenic compound.
For solid tumor or other cancer applications of the present methods
and devices, the test reagents can include a targeted
pharmaceutical agent such as, for example, antitumor monoclonal
antibodies, e.g. trastuzumab (Herceptin.RTM.), cetuximab
(Erbitux.RTM.), bevacizumab (Avastin.RTM.) and rituximab
(Rituxan.RTM.& or Mabthera.RTM.), and small molecule inhibitors
e.g., gefitinib (Iressa.RTM.), or erlotinib (Tarceva.RTM.) or
cytotoxic chemotherapy agents, such as, for example taxanes
(Taxotere.RTM.), antimetabolites (fluorouracil), alkylating agents,
platinum agents or anthracyclines. These exemplary pharmaceutical
agents can be used individually, in any combination with another
pharmaceutical agent disclosed herein, or in combination with
another compound.
After administering a test reagent, it can be determined if the
test reagent affects the expression of one or more markers, wherein
the presence, absence, or relative degree of such expression is
indicative of the susceptibility of the cells to a selected
pharmaceutical agent. These markers can include a wide array of ex
vivo biomarkers such as mRNA, a microRNA, cDNA, a protein, a
phosphoprotein, a posttranslational modification of a protein, or a
modification of histone or DNA packaging. For example, the marker
can be mRNA or cDNA for an early response gene (e.g., FOS or JUN)
associated with susceptibility to a pharmaceutical agent. The
presence, absence, or relative degree of expression of combinations
of markers in the presence of a test reagent can be indicative of
the susceptibility of the cells to a selected test reagent, such as
a pharmaceutical agent.
F. Sample Preparation and Stabilization
As described herein and illustrated in the figures, the cells
processed using the present invention can be prepared and
stabilized in a number of ways to permit a wide array of cellular
analyses to be performed on them. For example, the cells can be
prepared for nucleic acid analysis, protein analysis, and/or
analyzed using live cellular probes.
For nucleic acid analysis, a stabilizing reagent such as
RNAlater.RTM., RNA Protect Cell Reagent.RTM. (both available from
Qiagen), or ethanol can be added to the cells. The stabilized cells
can then be optionally lysed or have the nucleic acid of interest
otherwise extracted. The extracted and purified nucleic acid can
then be analyzed, for example, using PCR techniques.
In some embodiments, and as described above, the methods described
herein yield nucleic acids for further analysis. For these samples,
following dispersion and optional enrichment, the nucleic acids can
be stabilized or extracted (optionally) to yield high quality and
quantity nucleic acids. See, for example, Example 10 below and
FIGS. 13 and 14. This can be done, for example, by lysing the
desired cells following exposure to a test reagent and then
obtaining cDNA using reverse transcriptase and DNA primers. The DNA
primers can comprise nonspecific primer complementary to poly A,
e.g. oligo(dT).sub.12-18 or a specific primer complementary to a
mRNA transcript of interest. As one of skill in the art will
appreciate, the cells can be lysed using a variety of methods, such
as, chemical or mechanical means.
Optionally, the cells can be stabilized with reagents to detect
and/or preserve biomarker information, e.g., using reverse
transcriptase and DNA primer to obtain cDNA transcripts, preparing
RNA, DNA and protein for down stream molecular analysis.
For protein analysis, either whole cells or lysed cells can be
used. Intact whole cells can be fixed and stabilized with a
polymer, such as the one in Table 2 below, so that the sample
adheres to the isolated chamber, for example, a glass slide. These
samples can then be subjected to analysis, for example,
immunohistochemical (IHC) analysis. Lysed or otherwise ruptured
cells can be used in assays such as Western Blots and may not
require stabilization or fixation.
Slide preparation for morphological review by a pathologist and
protein analysis by IHC can be an output of the methods described
herein. Accordingly, the cells can also be prepared, optionally
using polymers, on glass slides for analysis of morphology and/or
immunohistochemistry. For example, the mixture disclosed in Table 2
can be used according to the example protocol in Table 3. See also
Maksem, J. A., V. Dhanwada, et al. (2006). "Testing automated
liquid-based cytology samples with a manual liquid-based cytology
method using residual cell suspensions from 500 ThinPrep cases."
Diagn. Cytopathol 34(6): 391-6).
TABLE-US-00002 TABLE 2 Polymer solution Agarose 0.18 g PEG 4.8 g
Alcohol Reagent 76.8 ml Poly L-lysine (0.1%) 0.25 ml Nonidet
P40**** 0.05 mL Total 240 mL
TABLE-US-00003 TABLE 3 Example Protocol 1 Dissolve 4.8 g of PEG in
15 ml of deionized water, heat up while stirring 2 Dissolve 0.18 g
of agarose in 15 ml of deionized water by heating the solution to
boiling 3 while maintaining vigorous mixing until the solution
optically clears 4 Immediately add the hot agarose solution to the
PEG solution 5 Dilute the solution with 133.2 ml of water (hot) and
cool to room temperature 6 Add 76.8 ml of reagent alcohol to the
solution with mixing 7 Adjust the final volume to 240 ml with
deionized water 8 Add 250 ul of poly-L-lysine solution 9 Add 50 ul
of IGEPAL CA-630 10 filter with cheese cloth, store at room
temperature for at least 72 hr before use
Live cellular probe analysis can involve adding a molecular probe
(such as MitoTracker.RTM. as described in the examples) at any
point in the method of processing the cells where the cells are
alive. This addition of the live cell probe should be made prior to
fixing or otherwise allowing the cells to die. For example, such a
probe can be added before or after cellular stabilization but prior
to cellular fixation.
In some embodiments, the cells can be stabilized and fixed by any
suitable means that will permit subsequent molecular analysis and
detection of markers. Generally, crosslinking fixatives such as
formalin are not preferred but may be present in small amounts that
will not interfere with subsequent analysis. Where the biomarker is
expression of a particular gene or genes, in one embodiment the
cells are lysed and exposed to reverse transcriptase and suitable
primers, so as to generate cDNA transcripts of mRNA transcripts in
the cells. This facilitates subsequent analysis, as cDNA is less
subject to degradation than mRNA.
In some embodiments, 1.times.10.sup.4 or more cells are processed
to stabilize any or all of the following: RNA, DNA, protein, and/or
phosphoproteins.
In some embodiments, the cells can be fixed after processing. Any
suitable means of fixation can be used, for example, air drying
techniques, adding a compound such as alcohol, e.g., a fixative
comprising a lower alkanol, e.g. methanol or ethanol, adding
formalin, adding an RNase inhibitor, adding agarose, adding
polyethylene glycol, adding poly 1-lysine, or adding one or more
chelator or antioxidant. In some embodiments, the fixative
comprises agarose, polyethylene glycol, octylphenoxy-polyethylene
glycol, poly-1-lysine, reagent alcohol and water.
A further embodiment of the methods of the present invention
includes a method for preparing solid tissue cells from a subject,
e.g., solid tumor cells from an animal or human subject having a
solid tumor, e.g., for determination of sensitivity of the cells to
a selected targeted pharmaceutical agent. An example method can
include the steps of: (a) obtaining solid tissue comprising desired
cells from the subject; (b) dispersing (e.g., using shear forces)
the tissue into single live cells and/or aggregates of not more
than 100 live cells, e.g., 10 to 100 cells; (c) enriching the
sample, e.g. removing contaminating materials from the live cells;
(d) distributing the live cells into test aliquots in isolated
chambers; (e) exposing the live cells to one or more test reagents;
and (f) treating the cells with a fixative and/or stabilizing agent
(e.g., an agent stabilizing RNA, DNA, proteins and/or
phosphoproteins) to fix the tumor cells and/or marker for further
analysis; wherein the fixation of the tumor cells and/or the marker
is completed within four hours of removal of the tissue from the
subject in an automated or manual fashion.
Another embodiment the invention provides a method of testing cells
wherein solid tumor cells are removed from a mammal (e.g., a human
patient), and while most of the cells, e.g., at least 65% of the
cells, e.g., at least 75% of the cells are viable and have not
replicated outside the body, exposing all or a portion of the cells
ex vivo to one or more test reagents, and stabilizing the cells,
optionally with a fixative (e.g., a polymer) that can preserve
biomarker information including cellular DNA, RNA, proteins, and/or
phosphoproteins. These biomarkers can be tested using molecular
analyses known to one of skill in the art or using the novel ex
vivo biomarker tests disclosed herein.
The following examples are further illustrative of the present
invention, but are not to be construed to limit the scope of the
present invention.
Example 1
Live Cell Processing
The importance of live cell processing has been demonstrated using
the live cell molecular probe, MitoTracker (available from
Invitrogen, Carlsbad, Calif.). MitoTracker localizes to
mitochondria when applied to living cells by passive diffusion
across the plasma membrane. The living cells were fixed to
stabilize the MitoTracker localization and analyzed by fluorescence
microscopy. Unlike currently available biopsy processing methods
utilizing methods, devices and systems according to the present
invention enables the study of live cells with molecular probes.
This is illustrated in FIGS. 20 and 21, where the specific
cytoplasmic localization of mitochondria (granular fluorescence,
left side--20A and 21A) was clearly demonstrated when the probe is
applied to live cells, but was uninformative when applied to cells
that were fixed using prior art methods (right side, 20B and
21B).
Example 2
MCF-7 and HCT-116 Dissaggregation Studies
MCF-7 (human breast carcinoma cells--ATCC#HTB-22) and HCT-116
(human colon carcinoma cells--ATCC#CCL-247) were used to examine
the impact of shear forces on cluster size, viability and cellular
activation in a semi-automated pipetting device. Briefly, MCF-7 and
HCT-116 cells were grown to 80% confluency in tissue culture then
removed from the plates by gently scraping with a rubber policeman
and suspended in growth medium to mimic the cell number and
fragment size in a typical FNA sample. One aliquot of the cell
suspension was passed through an automated pipetting apparatus
(Harvard Pipetter, Harvard Apparatus, Holliston, Mass.) with an 22
G needle four times (withdraw/infuse at 4.14 mL/min for each pass)
resulting in a wall shear stress exposure ranging from 100-800
dyne/cm.sup.2 and a total exposure time for each cell or aggregate
of 4 transits.times.14 msec/transit=56 msec. Representative samples
from each were cytocentrifuged onto a glass slide, fixed with 95%
ethanol and stained with the Papanicolaou stain. Photomicrographs
of representative areas were obtained (Magnification .times.200).
Note the decreasing cell cluster size with increasing shear forces.
These results are illustrated in FIG. 22.
Example 3
Aggregation Size Distribution
After using the method of example 2, average cluster size was then
quantified through the use of a non-flow imaging-based cell counter
that measures cell concentration and cell size distributions
(Cellometer.RTM., Nexcelom, Lawrence, Mass.). At 100 dyne/cm.sup.2
the average cluster size of MCF-7 cells was 97.+-.3 .mu.m and HCT
cells was 51.+-.6 .mu.m (FIG. 15). These data provide a range of
reproducible, optimal shear forces necessary to disperse aggregates
of live cells.
Example 4
Viability Analysis from Dispersion
After using the method of example 2, viability was also examined by
trypan blue exclusion assay at comparable shear forces from the
semi-automated pipetter. It was concluded that shear forces greater
than 800 dyne/cm.sup.2 resulted in more than a 40% decrease in
viability deemed too severe for live cell manipulations and
processing. See FIG. 16 for a graphical depiction of the results
obtained.
Example 5
Activation Analysis from Dispersion
A functional measurement of cellular activation includes FOS mRNA
induction determined by quantitative RT-PCR. FOS is an early
response gene associated with the EGFR pathway. In an experiment,
MCF-7 cells were grown in normal growth conditions in 6 well plates
to 80% confluency. Cells were gently scraped and exposed to
increasing shear forces (0-800 dyne/cm.sup.2 through the Harvard
Pipetter) in addition to increasing incubation times (0-45 minutes)
in the presence or absence of 100 ng/ml of EGF ligand (Sigma) prior
to RNA extraction. FOS mRNA induction peaks at 30-45 minutes and
returns to basal levels in approximately 60 minutes. FOS induction
is also stimulated as a result of incubation with EGF ligand.
Importantly, preliminary results indicate shear forces generated on
a semi-automated platform of 100-800 dyne/cm.sup.2 dispersion do
not result in significant cellular activation compared to EGF
stimulus. See FIGS. 17 and 18 for a graphical depiction of the
results obtained.
Example 6
Apparatus
FIG. 1 shows a schematic view of an example cartridge for use, for
example, in an apparatus, which provides a platform to integrate
the function of conducting disaggregation of tissue, the function
of cell-counter, the function of gene expression drug
susceptibility testing, and the function of fixing a sample for
further analysis. FIGS. 1-22 illustrate particular embodiments of
features described herein. Persons skilled in the art will
recognize how the various embodiments operate when the Figures are
considered in conjunction with the present description.
The apparatus comprises as its main components: a storage cartridge
1, which can be inserted into an apparatus or used for manual
processing, the cartridge having a plurality of small containers
in, for example, a 96-well plate format 2 removable mounted on the
cartridge, and a plurality of containers 3 for initial receipt,
dispersion and removal of contaminating materials from the samples.
The containers 3 may serve different functions, as depicted
schematically in FIG. 2 and previously described herein. The
cartridge has a label 4, which may be bar coded to facilitate
identification of the sample. Each of the containers comprises a
well and a seal that ensures biologic confinement of the contents
and is puncturable by a needle, but resealable upon removal of the
needle. A tissue sample is gathered from a patient, typically an
aspiration biopsy using a fine needle 5 by a physician, who
deposits the sample tissue into the receiver container 3 on the
cartridge 1.
The cartridge 1 is then slid into the apparatus and closing door
seals the apparatus, so that the cartridge 1 is biologically sealed
from the outside environment and sealed against release of any of
the biologically hazardous tissue sample. In an alternative
embodiment, the cartridge is a platform containing particular
reagents and can be processed manually.
The cartridge 1 can have one or more receptacles 6 for storage and
disposal of raw materials for use in conducting the manipulation of
the tissue sample. These raw materials include needle heads and
reagents. The needle head consists of a needle having an aperture
and a point and an annulus within the needle in fluid connection
with the aperture and a syringe. The apparatus is self-contained
with the exception of electric current, which can be supplied via
cord if necessary.
The cartridges can include a receiver container 2 that houses a
processing assembly, typically for mixing the tissue specimen
therein. The receiver container 3 is prepackaged with a
disaggregation solution of buffered saline; optionally further
comprising chelators, antioxidants, and viscosity modifiers, with
the constraint that the disaggregation solution should avoid the
use of proteases.
The receiver container 3 consists of a seal cover for the well.
This self-contained well and processing assembly arrangement
minimizes human operator exposure to biohazards. An engagement
extension protrudes through the seal cover.
The apparatus is fitted with an operator member, able to
selectively pick up a needle head from storage well 6 on cartridge
1, to operate a syringe of the needle head, and to operate certain
devices in the apparatus, such as the processing assembly on
receiver container.
In the first operation within apparatus, the operator member
retrieves a needle head from the storage well 6, moves to a
position relative to the receiver container 3 in which the sample
tissue has been deposited by the physician and which is prepackaged
with a disaggregation solution, such buffers, chelators and
antioxidant, punctures the seal on the receiver container 3 with
the needle head and submerses the needle head point in the
homogeneous solution mixture, withdraws a portion of the sample
from the receiver container 3 into the syringe of the needle head,
moves the needle head point to predetermined position within the
receiver container and dispenses the withdrawn sample in the same
or second receiver container 3 to disaggregate the tissue sample
into a homogeneous solution of intact tumor cells and contaminant
materials. This step is repeated as many times as necessary to
achieve the predetermined level of disaggregation of the tissue
sample. See, FIG. 3, e.g., 309.
The operator member (or operator if manually manipulated) retrieves
a new needle head from the cartridge storage well 6, moves to a
position relative to the receiver containers 3, punctures the seal
on selected receiver container 3 with the needle head and submerses
the needle head point in the homogeneous solution mixture,
withdraws a portion of the sample from the receiver container into
the syringe of the needle head, removes the needle from the
receiver container, moves to a position relative to a matrix
container, punctures the seal on the matrix container with the
needle head and deposits the sample portion from the syringe into
the matrix container 3. See also FIG. 2. The matrix container 3
consists of a loading chamber located above a bed of resin beads
which is supported above a collection chamber at the bottom of the
matrix container. The bed of resin is fitted with a plug that
extends from the top surface of the bed to the bottom surface of
the bed. The plug is constructed of material that permits
puncturing by a needle head at the top surface of bed and permits
the needle to extend through the bottom surface and into the
collection chamber. See also FIG. 2. Alternatively, the bed of
resin is fitted with a conduit extending from near the top of the
loading chamber through the bed of resin to near the bottom of the
collection chamber. In operation, the deposited sample solution
would be deposited above the resin bed, would gravity feed and also
be drawn though the bed of resin which removes the contaminants
from the solution and collects in the collection chamber. The top
of the conduit is situated so as to prevent any solution from
bypassing the resin bed before collecting in the collection
chamber.
In one embodiment, the sample is checked to ensure adequate numbers
of cells dispersed in the sample. The operator member retrieves a
needle head from the cartridge storage well 6, moves to a position
relative to the receiver container, punctures the seal on the
receiver container with the needle head and submerses the needle
head point in the homogeneous solution mixture, withdraws a
predetermined portion of the sample from the receiver container 3
into the syringe of the needle head, removes the needle head from
the receiver container 3, moves to a position relative to a cell
counter module and deposits the predetermined portion of the sample
in the counter module. The needle head is then withdrawn from the
counter container and disposed of in a disposal container on the
cartridge.
The counter module, which may be one of the receiver modules 3 on
the cartridge, is prepackaged with a dry dye on the interior of the
holding chamber of the counter container. Within the counter
container, the counter sample portion that has been deposited in
the holding chamber dissolves the dye, which in turn stains the
tumor cells. The predetermined sample portion funnels down into the
counter tube, which is optically scanned for cell count by a
scanner. The counter sample portion collects below the counter tube
in a holding well. The optical scanner relays the count to an
indicator that determines whether the count meets or exceeds the
predetermined count size indicating a successful biopsy sample. The
results of the analysis can then be displayed on a display and/or
printed by a printer. The results then immediately guide the
physician as to whether an additional biopsy is necessary. Provided
the sample is adequate, the patient is excused.
Once the cells are dispersed and contaminants removed, the cells
are divided into aliquots to be placed in a test matrix 2,
typically a 96 well plate format. Typically, the wells have been
prefilled with the desired media (buffered saline solution and test
reagents). The operator member picks up another needle from the
cartridge, remove the desired amount of dispersed and
decontaminated cells from the receiver container 3, moves to a
position relative to a series of sealed sample wells 2,
sequentially punctures the seal on each of the well, deposits a
predetermined amount of sample portion into each of the wells and
removes the needle head. The needle head is disposed of in the
disposal container 6.
Preferably, the wells 2 are prepackaged with various doses of
various pharmaceutical agents to test the susceptibility of the
tumor cells to each of the dosages and agents. Conditions for
maintaining the wells should be close to physiological conditions.
The pH of the medium in the wells should be close to physiological
pH, preferably between pH 6-8, more preferably between about pH 7
to 7.8, with pH 7.4 being most preferred. Physiological
temperatures range between about 30.degree. C. and 40.degree. C.
Cells are preferably maintained at temperatures between about
35.degree. C. and about 37.degree. C. Similarly, cells may be
cultured in levels of O.sub.2 that are comparatively reduced
relative to O.sub.2 concentrations in air, such that the O.sub.2
concentration is comparable to physiological levels (1-6%), rather
than 20% O.sub.2 in air. Given the short incubation times, it is
not generally necessary to oxygenate the cells.
After incubation with the agents for a predetermined length of
time, each well is treated with a fixative agent that fixes the
cells and the indicator agent for later analysis. This treatment is
accomplished with the operator member, a new needle head and a vial
of fixative agent.
Solid tumor cells can also be cryopreserved until they are needed,
by any method known in the art. The cells can be suspended in an
isotonic solution, preferably a cell culture medium, containing a
particular cryopreservant. Such cryopreservants include dimethyl
sulfoxide (DMSO), glycerol and the like. These cryopreservants are
used at a concentration of 5-15%, preferably 8-10%. Cells are
frozen gradually to a temperature of -10.degree. C. to -150.degree.
C., preferably -20.degree. C. to -100.degree. C., and more
preferably -150.degree. C.
It is clear, however, that modifications and/or additions can be
made to the apparatus 10 and method as described heretofore,
without departing from the field and scope of the present
invention. For example, the counter container 46 and optical
scanner 56 can be utilized on samples taken directly from the
receiver container 14 prior to the operation of the process
assembly 34.
Example 7
Dissaggregation Studies
MCF-7 human breast carcinoma cells (ATCC#HTB-22) are grown to 80%
confluency in tissue culture then removed from the plates by gently
scraping with a rubber policeman and suspended in growth medium.
One aliquot of the cell suspension is passed through an 18 G needle
twice (withdraw/infuse at 1 mL/s for each pass) resulting in a wall
shear stress exposure of 172 dyne/cm.sup.2 and a total exposure
time for each cell or aggregate of 4 transits.times.14
msec/transit=56 msec. A second aliquot is passed through an 18 G
needle five times (exposure time of 10 transits.times.14
msec/transit=140 msec). Representative samples from each are
cytocentrifuged onto a glass slides, fixed with 95% ethanol and
stained with the Papanicolaou stain. Photomicrographs of
representative areas are obtained (Magnification .times.200). For
comparison, another image is obtained from an ultrasound-guided
fine needle aspiration biopsy (FNA) of an enlarged lymph node found
to contain metastatic breast cancer. Another image from this same
human sample showed several groups of breast carcinoma cells in a
background of numerous lymphocytes Scraped MCF-7 and processed
MCF-7 with 2 withdraw/infuse cycles or 5 cycles through a 18 G
needle (1'') at 1 mL/sec. Note that 5 cycles of withdraw/infuse in
sample C is sufficient to result in lysis and released nuclei, a
condition that is not desired.
In the above experiment, an exposure of 2 cycles of withdrawal and
infusion of MCF-7 cells represents a preliminary estimate of an
operating condition, in the absence of EDTA, to disperse the
scraped MCF-7 monolayer. This experimental condition provides an
integrated shear exposure of t.sub.w(4.sub.transit)=172
dyne/cm.sup.2.times.0.056 sec=9.6 dyne-sec/cm.sup.2.
Example 8
Relative FOS Expression as Early Marker for Susceptibility to EGFR
Blockers
At least 35 targeted cancer drugs aimed at the epidermal growth
factor receptor (EGFR) are approved or in clinical trials.
Unfortunately, biomarkers predicting tumor sensitivity to EGFR
antagonists are unknown for most cancers. The variation in the
expression of the early response gene FOS as a distal effect of
EGFR inhibition can be evaluated and its relationship to antitumor
effects the growth-inhibitory and FOS-modulating effects of
gefitinib and erlotinib in human cancer cell lines (A431, CAL27,
HN11, HuCCT1, and Hep2) determined. Next, these cell lines can be
xenografted in mice and treated for 14 days with gefitinib (A431
and HuCCT1) or erlotinib (CAL27, HN11, and Hep2). Fine needle
aspiration biopsy of tumors is done at baseline and after 14 days
of therapy for FOS assessment. In addition, the feasibility of
analyzing this marker in five paired tumor samples from a clinical
trial of gefitinib in patients with solid tumors can be tested. In
culture, gefitinib and erlotinib decrease FOS mRNA levels in the
susceptible cell lines A431, CAL27, and HN11. Gefitinib or
erlotinib abrogate the increase in FOS expression in vivo in
EGFR-sensitive A431, CAL27, and HN11 tumors but not in resistant
strains. In summary, variations in FOS expression reflect the
pharmacologic actions of EGFR inhibitors with in vitro and in vivo
models. See, e.g., Jimeno A, Kulesza P, Kincaid E, Bouaroud N, Chan
A, Forastiere A, Brahmer J, Clark D P, Hidalgo M: C-fos Assessment
as a Marker of Anti-Epidermal Growth Factor Receptor Effect, Cancer
Res 2006, 66:2385-2390.
Example 9
Optimize Conditions for Rapid but Gentle Dispersion of FNAs
Selection of MCF-7 as Analog of Breast Tumor Cells in FNA
MCF-7 human breast cancer cells have been studied extensively as a
model for hormone dependent breast cancer. The cells are a
well-characterized estrogen receptor (ER) positive cell line and
therefore are a useful in-vitro model of breast cancer research.
The stable epithelial cell line is derived from primary culture of
human breast carcinoma cells obtained from a pleural effusion from
a female patient with metastatic disease (Soule, 1973). Since then
the MCF7 cell line has arguably become the most widely investigated
breast cancer model with thousands of citations as result of the
comparable clinical attributes. Similar to hormone dependent
ER-positive breast cancer, MCF7 cells are initially sensitive to
anti-estrogens such as tamoxifen and fulvestrant. The MCF7 cell
line has also served as the parental cell line for derivations of
numerous other breast cancer models, which have repeatedly
predicting clinical trial outcomes. Additionally, derivatives of
the MCF7 cell line have provided insight into the mechanisms of
resistance associated with first line hormonal therapy.
MCF-7 human breast carcinoma cells (ATCC#HTB-22) are grown in
Modified Improved Minimum Essential medium (Invitrogen, Carlsbad,
Calif.), 10% fetal bovine serum (Hyclone, Logan, Utah) and 1%
penicillin/streptomycin solution (10,000 IU ea., Invitrogen). Upon
80% confluency cells are washed once with 10 mL of DPBS and removed
from the flask by gentle scraping with a rubber cell scraper. Cells
are suspended in 10 mL of growth medium and divided into replicate
aliquots. This protocol has been validated to produce very large
aggregates that are a surrogate of tumor cell clusters present in
human breast cancer FNAs. The first aliquot (3 mL of suspended
cells) is imaged and sized by image analysis and a coulter counter.
After the initial size state of the first aliquot is counted, the
sample is trypsinized and all cells counted.
Selection of HCT-116 Colon Carcinoma Cell Line as Analog of
Metastatic Colon Carcinoma FNA
The human colon carcinoma cell line HCT-116 (ATCC# CCL-247) is
initially derived from a human male colon adenocarcinoma and has
been widely utilized in subsequent studies. Its morphology
resembles that of metastatic colon carcinoma and it has genetic
features common to human colon carcinomas, including a mutation in
codon 13 of the ras protooncogene. It is included in the NCI-60
panel of human cancer cell lines screened for pharmaceutical
sensitivity. Extensive cDNA microarray gene expression data and
correlative drug activity data are available on this cell line
(http://discover.nci.nih.gov/).
HCT-116 Culture: HCT-116 cells are propagated in McCoy's 5a
Modified Medium with 10% fetal bovine serum and incubated at 37 C
plus 5% CO2. Medium is renewed every 2-3 days and cells are split
when confluent at a subculture ratio of approximately 1:8 using a
trypsin-EDTA solution.
FOS Staining Method: Cell clusters are fixed by incubating the
slides in a solution containing 2% paraformaldehyde, 0.5% Triton
X-100 at 4.degree. C. for 15 minutes. The slides are then washed
with 3% BSA, 0.5% Triton X-100 in PBS and incubated with 50 .mu.l
of sheep polyclonal antibody against FOS (Cambridge Research Inc.,
Wilmington, Del.) at a dilution of 1:20 (3% BSA, 0.5% Triton in
PBS) for 2 hr. The slides are then washed three times with 5 ml of
3% BSA, 0.5% Triton in PBS solution. Each slide is incubated with
50 .mu.l of fluorescein donkey antisheep IgG (H+ L) conjugate
(Molecular Probes Inc., Eugene, Oreg.) (1:20 dilution) for 1 hr,
washed 4 times with PBS, and imaged.
Trypan Blue Exclusion
This simple test measures the ability of cells to exclude dye if
their membranes are intact. Depending on intensity, shear exposures
can transiently permeabilize membranes or perrnanently damage the
plasmalemma of cells. After dispersion experiments, cells will be
suspended in Hank's balanced salt solution. A total of 0.2 mL of
suspension is added to 0.8 mL of staining solution (0.5 mL of
sterile Trypan blue solution 0.4% (Sigma T-8154) in 0.3 mL HBSS),
incubated for 10 min, and 10 uL of the solution is counted with a
hemacytometer to obtain cell number and % dead cells.
Live/Dead Staining
While trypan blue exclusion is simple and accurate, the use of
fluorescent dyes is tested since a fluorescence determination of
cell viability and cell number is more readily automated and
miniaturized. Live/dead fluorescent staining uses two dyes: calcein
AM and ethidium homodimer (EthD-1). Calcein AM is a
non-fluorescent, cell permeable dye. It is cleaved to a fluorescent
form in live cells by intracellular esterases. Ethidium homodimer
(EthD-1) binds DNA and is a chromosome counter stain, but does not
penetrate live cells and can be used to detect dead cells. Standard
kits are available from ActiveMotif.
Detection of Apoptosis
Depending on the intensity of exposure, fluid shear forces can
cause necrosis or apoptosis. To measure apoptosis in non-fixed
cells at times of 0.5 to 1 hr after implementation of
disaggregation protocols, cell permeable NucView-488 caspase 3
substrate available from Biotium, which takes advantage of the high
DEVDase activity of caspase 3, is used. Caspase 3 is a common
marker of apoptosis. NucView.TM. 488 caspase 3 substrate is a
membrane permeable conjugate of a fluorogenic DNA dye and DEVD
substrate. Cleavage of the dye by intracellular caspase 3 releases
the DNA dye for simultaneous staining of the nucleus.
Determination of Fragmentation Rates from Experimental Data A
genetic algorithm (GA) is used for the purpose of regressing
size-dependent fragmentation kernels from a time series of
experimentally measured size distributions at t.sub.1, t.sub.2,
t.sub.3, t.sub.4, and t.sub.5 obtained after each withdraw/infusion
cycle of the experiment. The GA evolves an initial random
population of kernel models in accordance with the principles of
microevolution (crossover, random, fitness-mediated selection).
After each transit through a syringe, a size distribution is
measured at a discrete time into the fragmentation. For data
obtained at a given average tube shear rate (G.sub.avg), Equation 1
(a set of k ODEs) will be regressed by evaluation of the fitness of
test "chromosomes" each containing an evolvable parameter set
[A(i,G.sub.avg), y, g] for a.sub.i=A*(G.sub.avg).sup.y
(R.sub.hyd).sup.g for each cluster of i-cells. Note A(i,G.sub.avg)
is cell line and buffer-dependent.
Scraped monolayers of MCF-7 and HCT-116 are subjected to parabolic
shear fields in sterile syringe needles (wall shear stress from 5
to 500 dyne/cm.sup.2) for various times from 10 msec to 100 sec to
evaluate dispersion characteristics. Extensional flows are tested
via impinging flows with gap separations ranging from 100 microns
to 1000 microns. Cells are tested for viability. Size distributions
are obtained with a Coulter Counter. Cellular activation will be
measured with FOS immunostaining, an early response gene that can
be rapidly upregulated by high levels of shear forces. FOS
induction results in intense nuclear staining and can be scored as
% activation based on counting nuclei. Various buffers are tested
including those containing chelators and viscosity modifiers and
cell protectants, with the constraint of avoiding the use of
proteases that destroy potentially important cell surface
proteins.
FOS staining and viability in MCF-7 monolayers are assessed and
compared to those obtained for the scraped monolayers (FNA analog)
to establish baseline activation prior to shear disruption. To some
extent, this mimics activation during FNA acquisition prior to
dispersion. For each cell line, the most important parameters to
determine are the minimum shear exposure strength (wall shear
stress) and minimum shear exposure time (cumulative transit time
through the needle) to disaggregate the sample. An aliquot (0.2 to
3 mL of suspension of scraped cells) will be passed through 15 to
21 G needles (0.5 to 2-in. long) at flow rates from 0.1 to 5 mL/s
using a computer-controlled Harvard syringe pump 1 to 10 cycles.
Each cycle (withdraw/infuse) of a 10 ml syringe is defined as one
cycle. Needles are obtained from Popper & Sons
(www.popperandsons.com), a custom manufacturer of components for
automated liquid handling systems. Samples are imaged after each
cycle. By use of different gauge needles and different needle
lengths and different flow rates, the wall shear stress and
cumulative exposure time can be varied independently. The wall
shear stress scales linearly with flow rate Q but scales with the
third power of the radius.
Exposures to laminar wall shear stresses of 10 to 150 dyne/cm.sup.2
for times between 10 msec and 500 msec are generally sufficient to
control the disaggregation state of the sample to obtain clusters
of 5 to 10 cells/cluster. 2 to 4 cycles are generally optimum for
reliable dispersion of the scraped samples. For FOS activation and
cell viability studies, conditions and shear-induced FOS expression
are monitored to disrupt scraped monolayers so that <15% of
nuclei are positive for FOS expression.
Entry/Exit Effects
Experiments are conducted to evaluate the role of dispersal during
sample entry into and exit out of the syringe (where substantial
elongational flows can exist). Results are compared with the same
gauge needle and same flow rate, but different needle lengths
(0.5-in. versus 1.0-in. versus 2.0-in) and different cycle numbers
such that samples can be generated that are exposed to the same
wall shear stress and same cumulative shear exposure time, but
different numbers of entry/exit events. Generally, sample entry and
exit does not cause significant disaggregation.
For visual analysis of disaggregation after each cycle of
withdrawal/infusion, samples are placed on ice for immediate
cytocentrifugation. Cells will be prepared for microscopic analysis
by first centrifuging each sample at 2000 rpm in a tabletop
centrifuge (Hettich Rotina 46S) then resuspending the concentrated
cell pellet in 500 .mu.l of a balanced salt solution (Normosol).
The cells will then be applied to a glass microscope slide using a
cytocentrifuge at 750 rpm for 3 minutes (ThermoShandon Cytospin 4).
The slides will be immediately fixed in 95% ethanol and stained
with the Papanicolaou stain. Digital images of selected areas are
obtained using an Adobe Photoshop (v. 5.5) and a light microscope
(Olympus BX40) fitted with a digital camera (Kontron Elektronik
Prog/Res/3012). Cells are subjected to particle counting image
analysis (NIH Image) and results compared to cell counting obtained
for the original aliquot.
Impinging Flow Systems
Impinging flows can be reliably obtained by directing the end of
the needle toward a flat plate. Since the fluid jet exiting a
submerged tube rapidly decays within a few tube diameters, it is
important that the gap separation S be scaled with the needle gauge
(Ga) such that S=k(Inner Diameter) are k=0.5 to 5. The gap
separation is controlled with a manual micrometer. If large
diameter needles are used, the wall shear stress drops rapidly (See
Table 1). By directing small gauge needles (Ga=10 to 14) toward
flat bottom wells and using lower flows (.about.0.1 mL/s) the tube
wall shear stress can be maintained at <1 dyne/cm.sup.2. In this
configuration, disaggregation results by control of the impinging
flow. Impinging flows allow cells to experience bursts of
elongational shear forces for very short periods of time
(microseconds). Impinging flows with aggregates may be more
"nonlinear" in that the threshold for dispersal may be near the
threshold. Tenacious structures (stromal tissue) in FNAs may
require an impinging flow followed by standard tube flow. This can
be easily achieved in an automated manner by use of a stepper motor
to control needle position relative to the bottom of the
container.
Cellular Activation Studies
After the first round of studies to determine fluidic conditions
that disrupt cellular samples, the cell clusters are analyzed for
membrane integrity (trypan blue staining and live/dead staining),
cellular apoptosis (cell permeable caspase 3 fluorogenic assay),
and cellular activation (FOS staining). As a positive control,
conditions are already known for MCF-7 that cause loss of cell
viability. Less than 5% of nuclei are FOS positive in scraped
monolayers. Shear conditions to disrupt scraped monolayers to small
clusters where FOS positive nuclei are <15%. For apoptosis
studies, cells are allowed to incubate for 0.5 to 1 hr after
dispersal to evaluate onset of apoptosis after dispersion.
Buffer Modifications
The dispersion buffer can be modified to enhance sample dispersion
and minimize cellular activation. Chelation of extracellular
calcium with EDTA facilitates disassembly of junctions holding
cells together. EDTA exposure (5 mM, pH 7.4) followed by
recalcification is tested as a cellular stimulant on its own.
Reactive oxygen generation during dispersion of scraped monolayers
may also result in cellular activation. N-acetyl-L-cysteine (NAC, 5
mM) may reduce FOS induction during cell dispersion. Finally, 0.2%
(w/v) pluronic F68 is a polymer additive that has displayed
cyto-protectant activity via cell membrane interactions in other
membrane systems.
Example 10
Quantities of Cells
For HCT-116 and MCF-7 cells dispersed as in the previous examples
using 400 dyne/cm2, nucleic acids were stabilized using Cell
Protect. DNA and RNA were extracted using Qiagen extraction
techniques. The number of cells necessary to obtain adequate RNA
and DNA levels for analysis was determined. RNA from the cells was
evaluated using RNA integrity evaluation (RIN), optical density
260/280, and total .mu.g RNA. This experiment confirms, for
example, that a sample size of greater than 100,000 cells is
adequate for DNA and RNA analysis:
TABLE-US-00004 Output Input RNA DNA Cell Number RIN 260/280 ug
260/280 ug 10,000 4.95 .+-. 1.77 1.19 .+-. 0.21 0.53 .+-. 0.06
11.71 .+-. 18.23 0.15 .+-. 0.42 100,000 5.80 .+-. 0.85 1.46 .+-.
0.10 1.08 .+-. 0.15 0.82 .+-. 2.62 0.39 .+-. 0.10 1,000,000 9.43
.+-. 0.64 1.98 .+-. 0.02 8.83 .+-. 3.78 2.15 .+-. 0.18 4.59 .+-.
1.07 10,000,000 9.97 .+-. 0.06 2.06 .+-. 0.01 41.73 .+-. 7.21 2.06
.+-. 0.05 17.41 .+-. 2.14
These results are also shown graphically in FIGS. 13 and 14.
Example 11
Cell Counting
For samples dispersed in the previous example (MCF-7 and HCT-116),
cell counting is tested and validated with 10 to 100 .mu.L cell
suspension aliquots delivered to imaging chambers. Image counts are
compared to both hemacytometer and Coulter counter scores. For the
analysis of clumps, various imaging-processing algorithms is
validated using samples that are divided into subsets for complete
dispersion using trypsin and single cell counting. A Coulter
counter can be used to get the size distribution through the use of
a channelizer.
Nuclei Counting Protocol and Image Processing
The advantage of fluorescence staining of the nucleus is that
nuclei are particularly large and discrete cellular objects that
are easily identified in monolayer culture and suspension cells. In
validating imaging methods for 1-step cell counting without
separation or rinsing, dyes are tested that meet the following
criteria: (1) can be applied to cells without the need of complex
fix/wash/stain/wash protocols; (2) are easily excited at
wavelengths available with low cost diodes or lasers; (3) produce a
high signal-to-background ratio; and (3) produce a rapid stain of
the cells, including membrane permeable SYTO-11 and SYTO-16
(Invitrogen). The uv-dyes (DAPI, Hoescht 33342) meet many of these
criteria but require uv source, a disadvantage in Phase II when
imaging systems must be miniaturized and economized. Cells are
incubated in 10 mM SYTO11 (S7573, 508 nm EX/527 nm EM) or 10 uM
SYTO-16 (S7578, 488 nm EX/527 nm EM) for up to 5 min prior to the
imaging in chamber slides (Lab-Tek.TM.Chamber Slides.TM., Nunc;
Culture area: 0.4 cm.sup.2/well: working volume ul). Digital images
are obtained using Adobe Photoshop (v. 5.5) and a microscope
(Olympus) fitted with a digital camera (Kontron Elektronik
Prog/Res/3012). Images are scored for nuclear counts by both visual
inspection and image analysis software (NIH Image). These scores
are compared to both hemacytometer and Coulter counter scores. The
goal is to develop a fast and accurate nuclear counting protocol
where 100 uL of the cellular suspension is added to 10 uL of
staining solution and then imaged with blue excitation/green
emission after 5 min incubation. Several variants are available in
the SYTO series if the two chosen are inadequate. Similarly, the
permeable nuclear stain CyTRAK Orange (Biostatus Ltd.; 488 nm
EX/615 nm EM) can be tested for these applications.
The accurate counting of nuclei in small clusters is considerably
more difficult than counting nuclei in cell monolayers or single
cell suspensions. Edge detection and more complicated backgrounds
may cause errors in the image analysis software. To ensure accuracy
and consistency, comparison is made with cell counts obtained by
full trysin/EDTA dispersion, to determine what inaccuracy exists as
a function of mean cluster size, cluster size distribution, and
cluster density/image area. If needed, size and intensity standards
are added to the suspension for establishment of the signal range,
background subtraction, thresholding, object detection, and
particle counting.
These examples illustrate possible embodiments of the present
invention. While the invention has been particularly shown and
described with reference to some embodiments thereof, it will be
understood by those skilled in the art that they have been
presented by way of example only, and not limitation, and various
changes in form and details can be made therein without departing
from the spirit and scope of the invention. Thus, the breadth and
scope of the present invention should not be limited by any of the
above-described exemplary embodiments, but should be defined only
in accordance with the following claims and their equivalents. Any
headings used herein are provided solely for organizational
purposes and are not intended to impart any division or meaning to
this document, unless specifically indicated.
All documents cited herein, including websites, journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued or foreign patents, or any other documents,
are each entirely incorporated by reference herein, including all
data, tables, figures, and text presented in the cited
document.
* * * * *
References